Targeted Therapeutic Lysosomal Enzyme Fusion Proteins and Uses Thereof

ABSTRACT

The present invention relates in general to therapeutic fusion proteins useful to treat lysosomal storage diseases and methods for treating such diseases. Exemplary therapeutic fusion proteins comprise a lysosomal enzyme, a lysosomal targeting moiety, e.g., an IGF-II peptide, and a spacer peptide. Also provided are compositions and methods for treating Mucopolysaccharidosis Type IIIB (Sanfilippo B Syndrome), comprising a targeted therapeutic fusion protein comprising alpha-N-acetylglucosaminidase (Naglu), a lysosomal targeting moiety, e.g., an IGF-II peptide, and a spacer peptide.

This application is a divisional of U.S. patent application Ser. No.14/883,211, filed Oct. 14, 2015, allowed, which is a divisional of U.S.patent application Ser. No. 14/092,336, filed Nov. 27, 2013, which isnow U.S. Pat. No. 9,376,480, issued on Jun. 28, 2016, which claims thepriority benefit of U.S. Provisional Application No. 61/730,378, filedNov. 27, 2012, and U.S. Provisional Application No. 61/788,968, filedMar. 15, 2013, each of the aforementioned applications are incorporatedherein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates in general to therapeutic fusion proteinsuseful to treat lyssomal storage diseases and methods for treating suchdiseases. Exemplary therapeutic fusion proteins comprise a lysosomalenzyme, a lysosomal targeting moiety, e.g., an IGF-II peptide, and aspacer peptide. It is contemplated that the lysosomal enzyme isalpha-N-acetylglucosaminidase (Naglu) and the disease isMucopolysaccharidosis Type IIIB (Sanfilippo B Syndrome).

BACKGROUND

Normally, mammalian lysosomal enzymes are synthesized in the cytosol andtraverse the ER where they are glycosylated with N-linked, high mannosetype carbohydrate. In the golgi, the high mannose carbohydrate ismodified on lysosomal enzymes by the addition of mannose-6-phosphate(M6P) which targets these proteins to the lysosome. The M6P-modifiedproteins are delivered to the lysosome via interaction with either oftwo M6P receptors. The most favorable form of modification is when twoM6Ps are added to a high mannose carbohydrate.

More than forty lysosomal storage diseases (LSDs) are caused, directlyor indirectly, by the absence of one or more lysosomal enzymes in thelysosome. Enzyme replacement therapy for LSDs is being actively pursued.Therapy generally requires that LSD proteins be taken up and deliveredto the lysosomes of a variety of cell types in an M6P-dependent fashion.One possible approach involves purifying an LSD protein and modifying itto incorporate a carbohydrate moiety with M6P. This modified materialmay be taken up by the cells more efficiently than unmodified LSDproteins due to interaction with M6P receptors on the cell surface.

The inventors of the present application have previously developed apeptide based targeting technology that allows more efficient deliveryof therapeutic enzymes to the lysosomes. This proprietary technology istermed Glycosylation Independent Lysosomal Targeting (GILT) because apeptide tag replaces M6P as the moiety targeting the lysosomes. Detailsof the GILT technology are described in U.S. Application PublicationNos. 2003-0082176, 2004-0006008, 2003-0072761, 2005-0281805,2005-0244400, and international publications WO 03/032913, WO 03/032727,WO 02/087510, WO 03/102583, WO 2005/078077, the disclosures of all ofwhich are hereby incorporated by reference.

SUMMARY OF THE INVENTION

The present invention provides further improved compositions and methodsfor efficient lysosomal targeting based on the GILT technology. Amongother things, the present invention provides methods and compositionsfor targeting lysosomal enzymes to lysosomes using lysosomal targetingpeptides. The present invention also provides methods and compositionsfor targeting lysosomal enzymes to lysosomes using a lysosomal targetingpeptide that has reduced or diminished binding affinity for the IGF-Ireceptor and/or reduced or diminished binding affinity for the insulinreceptor, and/or is resistant to furin cleavage. The present inventionalso provides lysosomal enzyme fusion proteins comprising a lysosomalenzyme and IGF-II and spacer peptides that provide for improvedproduction and uptake into lysosomes of the lysosomal enzyme fusionprotein. In certain embodiments, the lysosomal enzyme isalpha-N-acetylglucosaminidase (Naglu).

In one aspect, the invention provides a targeted therapeutic fusionprotein comprising a lysosomal enzyme, a peptide tag having an aminoacid sequence at least 70% identical to amino acids 8-67 of mature humanIGF-II and a spacer peptide between the lysosomal enzyme and the IGF-IIpeptide tag. In various embodiments, the spacer peptide comprises one ormore GGGPS (SEQ ID NO: 14) or GGGSP (SEQ ID NO: 15) amino acidsequences, and optionally further comprises one or more of (i) GAP (SEQID NO: 9), (ii) GGGGS (SEQ ID NO: 12), (iii) GGGS (SEQ ID NO: 16), (iv)AAAAS (SEQ ID NO: 17), (v) AAAS (SEQ ID NO: 18), (vi) PAPA (SEQ ID NO:19), (vii) TPAPA (SEQ ID NO: 20), (viii) AAAKE (SEQ ID NO: 21) or (ix)GGGGA (SEQ ID NO: 60).

Exemplary lysosomal enzymes contemplated herein include those set out inTable 1.

In various embodiments, the targeted therapeutic fusion proteincomprises an amino acid sequence at least 85% identical to a humanα-N-acetylglucosaminidase (Naglu) protein (FIG. 1, SEQ ID NO: 1), apeptide tag having an amino acid sequence at least 70% identical toamino acids 8-67 of mature human IGF-II and a spacer peptide locatedbetween the Naglu amino acid sequence and the IGF-II peptide tag. Invarious embodiments, the spacer comprises the amino acid sequence GAP(SEQ ID NO: 9), GPS (SEQ ID NO: 10), or GGS (SEQ ID NO: 11). In variousembodiments, the spacer sequence comprises amino acids Gly-Pro-Ser (GPS)(SEQ ID NO: 10) between the amino acids of mature human IGF-II and theamino acids of human Naglu.

In various embodiments, the spacer peptide comprises one or more GGGGS(SEQ ID NO: 12) or GGGS (SEQ ID NO: 16) amino acid sequences. In variousembodiments, the spacer peptide comprises one or more GGGPS (SEQ ID NO:14) or GGGSP (SEQ ID NO: 15) amino acid sequences. In variousembodiments, the spacer peptide comprises one or more AAAAS (SEQ ID NO:17) or AAAS (SEQ ID NO: 18) amino acid sequences. In variousembodiments, the spacer peptide comprises one or more PAPA (SEQ ID NO:19) or TPAPA (SEQ ID NO: 20) amino acid sequences. In variousembodiments, the spacer peptide comprises one or more AAAKE (SEQ ID NO:21) amino acid sequences. In various embodiments, the spacer peptidecomprises one or more GGGGA (SEQ ID NO: 60) amino acid sequences.

In various embodiments, the spacer peptide comprises an amino acidsequence selected from the group consisting of: (GGGGS)_(n) (SEQ ID NOs:12, 56, 58, 91-94), (GGGGS)_(n)-GGGPS (SEQ ID NOs: 36, 95-100),GAP-(GGGGS)_(n)-GGGPS (SEQ ID NOs: 101-107), GAP-(GGGGS)_(n)-GGGPS-GAP(SEQ ID NOs: 37, 108-113), GAP-(GGGGS)_(n)-GGGPS-(GGGGS)_(n)-GAP (SEQ IDNOs: 114-162), GAP-GGGPS-(GGGGS)_(n)-GAP (SEQ ID NOs: 163-169),GAP-(GGGGS)_(n)-AAAAS-GGGPS-(GGGGS)_(n)-AAAA-GAP (SEQ ID NOs: 170-218),GAP-(GGGGS)_(n)-PAPAP-(Xaa)_(n)-GAP (SEQ ID NOs: 219-267),GAP-(GGGGS)_(n)-PAPAPT-(Xaa)_(n)-GAP (SEQ ID NOs: 268-316),GAP-(GGGGS)_(n)-(Xaa)n-PAPAP-(Xaa)n-(AAAKE)n-(Xaa)n-(GGGGS)_(n)-GAP (SEQID NOs: 544-551), (GGGGA)_(n) (SEQ ID NOs: 60, 79, 81, 317-320),(GGGGA)_(n)-GGGPS (SEQ ID NOs: 321-326), GAP-(GGGGA)_(n)-GGGPS (SEQ IDNOs: 327-333), GAP-(GGGGA)_(n)-GGGPS-GAP (SEQ ID NOs: 334-340),GAP-(GGGGA)_(n)-GGGPS-(GGGGA)_(n)-GAP (SEQ ID NOs: 341-389),GAP-GGGPS-(GGGGA)_(n)-GAP (SEQ ID NOs: 390-396),GAP-(GGGGA)_(n)-AAAAS-GGGPS-(GGGGA)-_(n)-AAAA-GAP (SEQ ID NOs: 397-445),GAP-(GGGGA)_(n)-PAPAP-(Xaa)_(n)-GAP (SEQ ID NOs: 446-494),GAP-(GGGGA)_(n)-PAPAPT-(Xaa)_(n)-GAP (SEQ ID NOs: 495-543),GAP-(GGGGA)_(n)-(Xaa)n-PAPAP-(Xaa)_(n)-(AAAKE)n-(Xaa)_(n)-(GGGGA)_(n)-GAP(SEQ ID NOs: 552-559); wherein n is 1 to 7. In various embodiments, n is1 to 4.

In various embodiments, the present invention provides an IGF-II peptidefor use as a peptide tag for targeting the peptide or fusion proteincomprising the peptide to a mammalian lysosome. In various embodiments,the present invention provides an IGF-II mutein. In various embodiments,the invention provides a furin-resistant IGF-II mutein having an aminoacid sequence at least 70% identical to mature human IGF-II(AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLET YCATPAKSE)(SEQ ID NO: 5) and a mutation that abolishes at least one furin proteasecleavage site.

In some embodiments, the present invention provides an IGF-II muteincomprising an amino acid sequence at least 70% identical to mature humanIGF-II. In various embodiments, the IGF-II mutein peptide tag comprisesamino acids 8-67 of mature human IGF-II. In various embodiments, theIGF-II mutein comprises a mutation that reduces or diminishes thebinding affinity for the insulin receptor as compared to the wild-typehuman IGF-II.

In some embodiments, the IGF-II mutein has diminished binding affinityfor the IGF-I receptor relative to the affinity of naturally-occurringhuman IGF-II for the IGF-I receptor.

In various embodiments, the present invention provides a targetedtherapeutic fusion protein containing a lysosomal enzyme; and an IGF-IImutein having an amino acid sequence at least 70% identical to maturehuman IGF-II, wherein the IGF-II mutein is resistant to furin cleavageand binds to the human cation-independent mannose-6-phosphate receptorin a mannose-6-phosphate-independent manner.

In some embodiments, the present invention provides a targetedtherapeutic fusion protein containing a lysosomal enzyme; and an IGF-IImutein having an amino acid sequence at least 70% identical to maturehuman IGF-II, and having diminished binding affinity for the insulinreceptor relative to the affinity of naturally-occurring human IGF-IIfor the insulin receptor. In a related embodiment, the IGF-II mutein isresistant to furin cleavage and binds to the human cation-independentmannose-6-phosphate receptor in a mannose-6-phosphate-independentmanner.

In various embodiments, an IGF-II mutein suitable for the inventionincludes a mutation within a region corresponding to amino acids 30-40of mature human IGF-II. In some embodiments, an IGF-II mutein suitablefor the invention includes a mutation within a region corresponding toamino acids 34-40 of mature human IGF-II such that the mutationabolishes at least one furin protease cleavage site. In someembodiments, a suitable mutation is an amino acid substitution, deletionand/or insertion. In some embodiments, the mutation is an amino acidsubstitution at a position corresponding to Arg37 or Arg40 of maturehuman IGF-II. In some embodiments, the amino acid substitution is a Lysor Ala substitution.

In some embodiments, a suitable mutation is a deletion or replacement ofamino acid residues corresponding to positions selected from the groupconsisting of 30-40, 31-40, 32-40, 33-40, 34-40, 30-39, 31-39, 32-39,34-37, 33-39, 34-39, 35-39, 36-39, 37-40 of mature human IGF-II, andcombinations thereof.

In various embodiments, an IGF-II mutein according to the inventionfurther contains a deletion or a replacement of amino acidscorresponding to positions 2-7 of mature human IGF-II. In variousembodiments, an IGF-II mutein according to the invention furtherincludes a deletion or a replacement of amino acids corresponding topositions 1-7 of mature human IGF-II. In various embodiments, an IGF-IImutein according to the invention further contains a deletion or areplacement of amino acids corresponding to positions 62-67 of maturehuman IGF-II. In various embodiments, an IGF-II mutein according to theinvention further contains an amino acid substitution at a positioncorresponding to Tyr27, Leu43, or Ser26 of mature human IGF-II. Invarious embodiments, an IGF-II mutein according to the inventioncontains at least an amino acid substitution selected from the groupconsisting of Tyr27Leu, Leu43Val, Ser26Phe and combinations thereof. Invarious embodiments, an IGF-II mutein according to the inventioncontains amino acids corresponding to positions 48-55 of mature humanIGF-II. In various embodiments, an IGF-II mutein according to theinvention contains at least three amino acids selected from the groupconsisting of amino acids corresponding to positions 8, 48, 49, 50, 54,and 55 of mature human IGF-II. In various embodiments, an IGF-II muteinof the invention contains, at positions corresponding to positions 54and 55 of mature human IGF-II, amino acids each of which is uncharged ornegatively charged at pH 7.4. In various embodiments, the IGF-II muteinhas diminished binding affinity for the IGF-I receptor relative to theaffinity of naturally-occurring human IGF-II for the IGF-I receptor. Invarious embodiments, the IGF-II mutein is IGF2 Δ8-67 R37A (i.e., aminoacids 8-67 of mature human IGF-II with the Arg at position 37 of maturehuman IGF-II substituted by Ala).

In various embodiments, the peptide tag is attached to the N-terminus orC-terminus of the lysosomal enzyme, therefore is an N-terminal tag or aC-terminal tag, respectively. In various embodiments, the peptide tag isa C-terminal tag.

In some embodiments, a lysosomal enzyme suitable for the invention ishuman alpha-N-acetylglucosaminidase (Naglu) (FIG. 1), or a functionalfragment or variant thereof. In some embodiments, a lysosomal enzymesuitable for the invention includes amino acids 1-743 of humanalpha-N-acetylglucosaminidase or amino acids 24-743 of humanalpha-N-acetylglucosaminidase, which lacks a signal sequence.

In various embodiments, a targeted therapeutic fusion protein of theinvention further includes a spacer between the lysosomal enzyme and theIGF-II mutein.

In various embodiments, the spacer comprises an alpha-helical structureor a rigid structure.

In various embodiments, the spacer comprises one or more Gly-Ala-Pro(GAP) (SEQ ID NO: 9), Gly-Pro-Ser (GPS) (SEQ ID NO: 10), or Gly-Gly-Ser(GGS) (SEQ ID NO: 11) amino acid sequences.

In some embodiments, the spacer is selected from the group consisting of

(SEQ ID NO: 22) EFGGGGSTR, (SEQ ID NO: 9) GAP, (SEQ ID NO: 12) GGGGS,(SEQ ID NO: 23) GPSGSPG, (SEQ ID NO: 24) GPSGSPGT, (SEQ ID NO: 25)GPSGSPGH, (SEQ ID NO: 26) GGGGSGGGGSGGGGSGGGGSGGGPST, (SEQ ID NO: 27)GGGGSGGGGSGGGGSGGGGSGGGPSH, (SEQ ID NO: 28)GGGGSGGGGSGGGGSGGGGSGGGPSGGGGSGGGPS, (SEQ ID NO: 29)GAPGGGGSGGGGSGGGGSGGGGSGGGPSGGGGSGGGPSGAP, (SEQ ID NO: 30)GGGGSGGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGGSGGGGSGGGP S, (SEQ ID NO: 31)GAPGGGGSGGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGGSGGGGSGG GPSGAP,(SEQ ID NO: 32) GGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGPS, (SEQ ID NO: 33)GAPGGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGPSGAP, (SEQ ID NO: 34)GGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGPSGGGGSGGGGSGGGP S, (SEQ ID NO: 35)GAPGGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGPSGGGGSGGGGSGG GPSGAP,(SEQ ID NO: 36) GGGGSGGGGSGGGGSGGGGSGGGPS, (SEQ ID NO: 37)GAPGGGGSGGGGSGGGGSGGGGSGGGPSGAP, (SEQ ID NO: 38)GGGGSGGGGSAAAASGGGGSGGGPS, (SEQ ID NO: 39)GAPGGGGSGGGGSAAAASGGGGSGGGPSGAP, (SEQ ID NO: 40)GGGGSGGGGSAAAASGGGGSGGGGSAAAASGGGGSGGGGSAAAASGGGP S, (SEQ ID NO: 41)GAPGGGGSGGGGSAAAASGGGGSGGGGSAAAASGGGGSGGGGSAAAASGG GPSGAP,(SEQ ID NO: 42) GGGGSGGGGSAAAASGGGPSGGGGSAAAASGGGPSGGGGSAAAASGGGP S,(SEQ ID NO: 43) GAPGGGGSGGGGSAAAASGGGPSGGGGSAAAASGGGPSGGGGSAAAASGGGPSGAP, (SEQ ID NO: 44) GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPGPS,(SEQ ID NO: 45) GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPGPS GAP,(SEQ ID NO: 46) GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTGP S,(SEQ ID NO: 47) GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTGPSGAP, (SEQ ID NO: 48) GGGSPAPTPTPAPTPAPTPAGGGPS, (SEQ ID NO: 49)GAPGGGSPAPTPTPAPTPAPTPAGGGPSGAP, (SEQ ID NO: 50)GGGSPAPAPTPAPAPTPAPAGGGPS, (SEQ ID NO: 51)GAPGGGSPAPAPTPAPAPTPAPAGGGPSGAP, (SEQ ID NO: 52)GGGSAEAAAKEAAAKEAAAKAGGPS, (SEQ ID NO: 53)GAPGGGSAEAAAKEAAAKEAAAKAGGPSGAP, (SEQ ID NO: 54)GGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGG, (SEQ ID NO: 55)GAPGGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGGGAP, (SEQ ID NO: 56)GGGGSGGGGSGGGGS, (SEQ ID NO: 57) GAPGGGGSGGGGSGGGGSGAP, (SEQ ID NO: 58)GGGGSGGGGSGGGGSGGGGS, (SEQ ID NO: 59) GAPGGGGSGGGGSGGGGSGGGGSGAP,(SEQ ID NO: 60) GGGGA, (SEQ ID NO: 61) GGGGAGGGGAGGGGAGGGGAGGGPST,(SEQ ID NO: 62) GGGGAGGGGAGGGGAGGGGAGGGPSH, (SEQ ID NO: 63)GGGGAGGGGAGGGGAGGGGAGGGPSGGGGAGGGPS, (SEQ ID NO: 64)GAPGGGGAGGGGAGGGGAGGGGAGGGPSGGGGAGGGPSGAP, (SEQ ID NO: 65)GGGGAGGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGGAGGGGAGGGP S, (SEQ ID NO: 66)GAPGGGGAGGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGGAGGGGAGG GPSGAP,(SEQ ID NO: 67) GGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGPS, (SEQ ID NO: 68)GAPGGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGPSGAP, (SEQ ID NO: 69)GGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGPSGGGGAGGGGAGGGP S, (SEQ ID NO: 70)GAPGGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGPSGGGGAGGGGAGG GPSGAP,(SEQ ID NO: 71) GGGGAGGGGAGGGGAGGGGAGGGPS, (SEQ ID NO: 72)GAPGGGGAGGGGAGGGGAGGGGAGGGPSGAP, (SEQ ID NO: 73)GGGGAGGGGAAAAASGGGGAGGGPS, (SEQ ID NO: 74)GAPGGGGAGGGGAAAAASGGGGAGGGPSGAP, (SEQ ID NO: 75)GGGGAGGGGAAAAASGGGGAGGGGAAAAASGGGGAGGGGAAAAASGGGP S, (SEQ ID NO: 76)GAPGGGGAGGGGAAAAASGGGGAGGGGAAAAASGGGGAGGGGAAAAASGG GPSGAP,(SEQ ID NO: 77) GGGGAGGGGAAAAASGGGPSGGGGAAAAASGGGPSGGGGAAAAASGGGP S,(SEQ ID NO: 78) GAPGGGGAGGGGAAAAASGGGPSGGGGAAAAASGGGPSGGGGAAAAASGGGPSGAP, (SEQ ID NO: 79) GGGGAGGGGAGGGGA, (SEQ ID NO: 80)GAPGGGGAGGGGAGGGGAGAP, (SEQ ID NO: 81) GGGGAGGGGAGGGGAGGGGA,(SEQ ID NO: 82) GAPGGGGAGGGGAGGGGAGGGGAGAP, (SEQ ID NO: 83)GGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGPS [or (GGGGA)₈GGGPS],(SEQ ID NO: 84) GGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGPSH[or (GGGGA)₈GGGPSH], (SEQ ID NO: 85)GGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGPS [or (GGGGA)₉GGGPS],(SEQ ID NO: 86) GGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGPSH [or (GGGGA)₉GGGPSH,] (SEQ ID NO: 87) GGGGPAPGPGPAPGPAPGPAGGGPS,(SEQ ID NO: 88) GAPGGGGPAPGPGPAPGPAPGPAGGGPGGAP, (SEQ ID NO: 89)GGGGPAPAPGPAPAPGPAPAGGGPS, and (SEQ ID NO: 90)GAPGGGGPAPAPGPAPAPGPAPAGGGPGGAP.

In some embodiments, the spacer is selected from the group consisting of

(SEQ ID NO: 36) GGGGSGGGGSGGGGSGGGGSGGGPS, (SEQ ID NO: 44)GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPGPS, (SEQ ID NO: 45)GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPG PSGAP, (SEQ ID NO: 46)GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTG PS, (SEQ ID NO: 47)GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT STGPSGAP,(SEQ ID NO: 48) GGGSPAPTPTPAPTPAPTPAGGGPS, (SEQ ID NO: 49)GAPGGGSPAPTPTPAPTPAPTPAGGGPSGAP, (SEQ ID NO: 50)GGGSPAPAPTPAPAPTPAPAGGGPS, (SEQ ID NO: 51)GAPGGGSPAPAPTPAPAPTPAPAGGGPSGAP, (SEQ ID NO: 52)GGGSAEAAAKEAAAKEAAAKAGGPS, (SEQ ID NO: 53)GAPGGGSAEAAAKEAAAKEAAAKAGGPSGAP, (SEQ ID NO: 54)GGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGG, (SEQ ID NO: 55)GAPGGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGGGAP, and (SEQ ID NO: 71)GGGGAGGGGAGGGGAGGGGAGGGPS.

In some embodiments, the spacer is selected from the group consisting of

(SEQ ID NO: 36) GGGGSGGGGSGGGGSGGGGSGGGPS, (SEQ ID NO: 47)GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST GPSGAP,(SEQ ID NO: 51) GAPGGGSPAPAPTPAPAPTPAPAGGGPSGAP, (SEQ ID NO: 55)GAPGGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGGGAP, and (SEQ ID NO: 71)GGGGAGGGGAGGGGAGGGGAGGGPS.

In various embodiments, the fusion protein further comprises apharmaceutically acceptable carrier, diluents or excipient.

The present invention also provides nucleic acids encoding the IGF-IImutein or the targeted therapeutic fusion protein as described invarious embodiments above. The present invention further providesvarious cells containing the nucleic acid of the invention.

The present invention provides pharmaceutical compositions suitable fortreating lysosomal storage disease containing a therapeuticallyeffective amount of a targeted therapeutic fusion protein of theinvention. The invention further provides methods of treating lysosomalstorage diseases comprising administering to a subject in need oftreatment a targeted therapeutic fusion protein according to theinvention. In some embodiments, the lysosomal storage disease isMucopolysaccharidosis Type IIIB (Sanfilippo B Syndrome).

In another aspect, the present invention provides a method of producinga targeted therapeutic fusion protein including a step of culturingmammalian cells in a cell culture medium, wherein the mammalian cellscarry the nucleic acid of the invention, in particular, as described invarious embodiments herein; and the culturing is performed underconditions that permit expression of the targeted therapeutic fusionprotein.

In yet another aspect, the present invention provides a method ofproducing a targeted therapeutic fusion protein including a step ofculturing furin-deficient cells (e.g., furin-deficient mammalian cells)in a cell culture medium, wherein the furin-deficient cells carry anucleic acid encoding a fusion protein comprising a lysosomal enzyme andan IGF-II mutein having an amino acid sequence at least 70% identical tomature human IGF-II, wherein the IGF-II mutein binds to the humancation-independent mannose-6-phosphate receptor in amannose-6-phosphate-independent manner; and wherein the culturing isperformed under conditions that permit expression of the targetedtherapeutic fusion protein.

In various embodiments, it is contemplated that certain of the targetedtherapeutic proteins comprising a spacer as described herein exhibitincreased expression of active protein when expressed recombinantlycompared to targeted therapeutic proteins comprising a different spacerpeptide. In various embodiments, it is also contemplated that targetedtherapeutic proteins described herein may have increased activitycompared to other targeted therapeutic proteins herein. It iscontemplated that those targeted therapeutic proteins exhibitingincreased expression of active protein and/or having increased activitycompared to other targeted therapeutic proteins comprising a differentspacer peptide are used for further experimentation.

In another aspect, the invention provides a method for treating alysosomal storage disease in a subject comprising administering to thesubject a therapeutically effective amount of a pharmaceuticalcomposition comprising a fusion protein comprising a lysosomal enzyme, apeptide tag having an amino acid sequence at least 70% identical toamino acids 8-67 of mature human IGF-II and a spacer peptide locatedbetween the lysosomal enzyme amino acid sequence and the IGF-II peptidetag. In various embodiments, the spacer peptide comprises one or moreGGGPS (SEQ ID NO: 14) or GGGSP (SEQ ID NO: 15) amino acid sequences, andoptionally further comprises one or more of (i) GAP (SEQ ID NO: 9), (ii)GGGGS (SEQ ID NO: 12), (iii) GGGS (SEQ ID NO: 16), (iv) AAAAS (SEQ IDNO: 17), (v) AAAS (SEQ ID NO: 18), (vi) PAPA (SEQ ID NO: 19), (vii)TPAPA (SEQ ID NO: 20), (viii) AAAKE (SEQ ID NO: 21) or (ix) GGGGA (SEQID NO: 60).

In various embodiments, the spacer peptide comprises an amino acidsequence selected from the group consisting of:

(SEQ ID NOs: 12, 56, 58, 91-94) (GGGGS)n, (SEQ ID NOs: 36, 95-100)(GGGGS)n-GGGPS, (SEQ ID NOs: 101-107) GAP-(GGGGS)n-GGGPS,(SEQ ID NOs: 37, 108-113) GAP-(GGGGS)n-GGGPS-GAP, (SEQ ID NOs: 114-162)GAP-(GGGGS)n-GGGPS-(GGGGS)n-GAP, (SEQ ID NOs: 163-169)GAP-GGGPS-(GGGGS)n-GAP, (SEQ ID NOs: 170-218)GAP-(GGGGS)n-AAAAS-GGGPS-(GGGGS)n-AAAA-GAP, (SEQ ID NOs: 219-267)GAP-(GGGGS)n-PAPAP-(Xaa)n-GAP, (SEQ ID NOs: 268-316)GAP-(GGGGS)n-PAPAPT-(Xaa)n-GAP, (SEQ ID NOs: 544-551)GAP-(GGGGS)n-(Xaa)n-PAPAP-(Xaa)n-(AAAKE)n-(Xaa)n- (GGGGS)n-GAP,(SEQ ID NOs: 60, 79, 81, 317-320) (GGGGA)n, (SEQ ID NOs: 321-326)(GGGGA)n-GGGPS, (SEQ ID NOs: 327-333) GAP-(GGGGA)n-GGGPS,(SEQ ID NOs: 334-340) GAP-(GGGGA)n-GGGPS-GAP, (SEQ ID NOs: 341-389)GAP-(GGGGA)n-GGGPS-(GGGGA)n-GAP, (SEQ ID NOs: 390-396)GAP-GGGPS-(GGGGA)n-GAP, (SEQ ID NOs: 397-445)GAP-(GGGGA)n-AAAAS-GGGPS-(GGGGA)n-AAAA-GAP, (SEQ ID NOs: 446-494)GAP-(GGGGA)n-PAPAP-(Xaa)n-GAP, (SEQ ID NOs: 495-543)GAP-(GGGGA)n-PAPAPT-(Xaa)n-GAP, (SEQ ID NOs: 552-559)GAP-(GGGGA)n-(Xaa)n-PAPAP-(Xaa)n-(AAAKE)n-(Xaa)n- (GGGGA)n-GAP;wherein n is 1 to 7, optionally n is 1 to 4.

In various embodiments, the spacer peptide has an amino acid sequenceselected from the group consisting of

(SEQ ID NO: 22) EFGGGGSTR, (SEQ ID NO: 9) GAP, (SEQ ID NO: 12) GGGGS,(SEQ ID NO: 23) GPSGSPG, (SEQ ID NO: 24) GPSGSPGT, (SEQ ID NO: 25)GPSGSPGH, (SEQ ID NO: 26) GGGGSGGGGSGGGGSGGGGSGGGPST, (SEQ ID NO: 27)GGGGSGGGGSGGGGSGGGGSGGGPSH, (SEQ ID NO: 28)GGGGSGGGGSGGGGSGGGGSGGGPSGGGGSGGGPS, (SEQ ID NO: 29)GAPGGGGSGGGGSGGGGSGGGGSGGGPSGGGGSGGGPSGAP, (SEQ ID NO: 30)GGGGSGGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGGSGGGGSGGGP S, (SEQ ID NO: 31)GAPGGGGSGGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGGSGGGGSGG GPSGAP,(SEQ ID NO: 32) GGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGPS, (SEQ ID NO: 33)GAPGGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGPSGAP, (SEQ ID NO: 34)GGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGPSGGGGSGGGGSGGGP S, (SEQ ID NO: 35)GAPGGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGPSGGGGSGGGGSGG GPSGAP,(SEQ ID NO: 36) GGGGSGGGGSGGGGSGGGGSGGGPS, (SEQ ID NO: 37)GAPGGGGSGGGGSGGGGSGGGGSGGGPSGAP, (SEQ ID NO: 38)GGGGSGGGGSAAAASGGGGSGGGPS, (SEQ ID NO: 39)GAPGGGGSGGGGSAAAASGGGGSGGGPSGAP, (SEQ ID NO: 40)GGGGSGGGGSAAAASGGGGSGGGGSAAAASGGGGSGGGGSAAAASGGGP S, (SEQ ID NO: 41)GAPGGGGSGGGGSAAAASGGGGSGGGGSAAAASGGGGSGGGGSAAAASGG GPSGAP,(SEQ ID NO: 42) GGGGSGGGGSAAAASGGGPSGGGGSAAAASGGGPSGGGGSAAAASGGGP S,(SEQ ID NO: 43) GAPGGGGSGGGGSAAAASGGGPSGGGGSAAAASGGGPSGGGGSAAAASGGGPSGAP, (SEQ ID NO: 44) GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPGPS,(SEQ ID NO: 45) GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPGPS GAP,(SEQ ID NO: 46) GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTGP S,(SEQ ID NO: 47) GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTGPSGAP, (SEQ ID NO: 48) GGGSPAPTPTPAPTPAPTPAGGGPS, (SEQ ID NO: 49)GAPGGGSPAPTPTPAPTPAPTPAGGGPSGAP, (SEQ ID NO: 50)GGGSPAPAPTPAPAPTPAPAGGGPS, (SEQ ID NO: 51)GAPGGGSPAPAPTPAPAPTPAPAGGGPSGAP, (SEQ ID NO: 52)GGGSAEAAAKEAAAKEAAAKAGGPS, (SEQ ID NO: 53)GAPGGGSAEAAAKEAAAKEAAAKAGGPSGAP, (SEQ ID NO: 54)GGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGG, (SEQ ID NO: 55)GAPGGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGGGAP, (SEQ ID NO: 56)GGGGSGGGGSGGGGS, (SEQ ID NO: 57) GAPGGGGSGGGGSGGGGSGAP, (SEQ ID NO: 58)GGGGSGGGGSGGGGSGGGGS, (SEQ ID NO: 59) GAPGGGGSGGGGSGGGGSGGGGSGAP,(SEQ ID NO: 60) GGGGA, (SEQ ID NO: 61) GGGGAGGGGAGGGGAGGGGAGGGPST,(SEQ ID NO: 62) GGGGAGGGGAGGGGAGGGGAGGGPSH, (SEQ ID NO: 63)GGGGAGGGGAGGGGAGGGGAGGGPSGGGGAGGGPS, (SEQ ID NO: 64)GAPGGGGAGGGGAGGGGAGGGGAGGGPSGGGGAGGGPSGAP, (SEQ ID NO: 65)GGGGAGGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGGAGGGGAGGGP S, (SEQ ID NO: 66)GAPGGGGAGGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGGAGGGGAGG GPSGAP,(SEQ ID NO: 67) GGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGPS, (SEQ ID NO: 68)GAPGGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGPSGAP, (SEQ ID NO: 69)GGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGPSGGGGAGGGGAGGGP S, (SEQ ID NO: 70)GAPGGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGPSGGGGAGGGGAGG GPSGAP,(SEQ ID NO: 71) GGGGAGGGGAGGGGAGGGGAGGGPS, (SEQ ID NO: 72)GAPGGGGAGGGGAGGGGAGGGGAGGGPSGAP, (SEQ ID NO: 73)GGGGAGGGGAAAAASGGGGAGGGPS, (SEQ ID NO: 74)GAPGGGGAGGGGAAAAASGGGGAGGGPSGAP, (SEQ ID NO: 75)GGGGAGGGGAAAAASGGGGAGGGGAAAAASGGGGAGGGGAAAAASGGGP S, (SEQ ID NO: 76)GAPGGGGAGGGGAAAAASGGGGAGGGGAAAAASGGGGAGGGGAAAAASGG GPSGAP,(SEQ ID NO: 77) GGGGAGGGGAAAAASGGGPSGGGGAAAAASGGGPSGGGGAAAAASGGGP S,(SEQ ID NO: 78) GAPGGGGAGGGGAAAAASGGGPSGGGGAAAAASGGGPSGGGGAAAAASGGGPSGAP, (SEQ ID NO: 79) GGGGAGGGGAGGGGA, (SEQ ID NO: 80)GAPGGGGAGGGGAGGGGAGAP, (SEQ ID NO: 81) GGGGAGGGGAGGGGAGGGGA,(SEQ ID NO: 82) GAPGGGGAGGGGAGGGGAGGGGAGAP, (SEQ ID NO: 83)GGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGPS [or (GGGGA)8GGGPS],(SEQ ID NO: 84) GGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGPSH[or (GGGGA)8GGGPSH], (SEQ ID NO: 85)GGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGPS [or (GGGGA)9GGGPS],(SEQ ID NO: 86) GGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGPSH [or (GGGGA)9GGGPSH,] (SEQ ID NO: 87) GGGGPAPGPGPAPGPAPGPAGGGPS,(SEQ ID NO: 88) GAPGGGGPAPGPGPAPGPAPGPAGGGPGGAP, (SEQ ID NO: 89)GGGGPAPAPGPAPAPGPAPAGGGPS, and (SEQ ID NO: 90)GAPGGGGPAPAPGPAPAPGPAPAGGGPGGAP.

In various embodiments, the spacer peptide has an amino acid sequenceselected from the group consisting of

(SEQ ID NO: 36) GGGGSGGGGSGGGGSGGGGSGGGPS, (SEQ ID NO: 44)GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPGPS, (SEQ ID NO: 45)GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPGPS GAP, (SEQ ID NO: 46)GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTGP S, (SEQ ID NO: 47)GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST GPSGAP,(SEQ ID NO: 48) GGGSPAPTPTPAPTPAPTPAGGGPS, (SEQ ID NO: 49)GAPGGGSPAPTPTPAPTPAPTPAGGGPSGAP, (SEQ ID NO: 50)GGGSPAPAPTPAPAPTPAPAGGGPS, (SEQ ID NO: 51)GAPGGGSPAPAPTPAPAPTPAPAGGGPSGAP, (SEQ ID NO: 52)GGGSAEAAAKEAAAKEAAAKAGGPS, (SEQ ID NO: 53)GAPGGGSAEAAAKEAAAKEAAAKAGGPSGAP, (SEQ ID NO: 54)GGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGG, (SEQ ID NO: 55)GAPGGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGGGAP, and (SEQ ID NO: 71)GGGGAGGGGAGGGGAGGGGAGGGPS.

In various embodiments, the spacer peptide has an amino acid sequenceselected from the group consisting of

(SEQ ID NO: 36) GGGGSGGGGSGGGGSGGGGSGGGPS, (SEQ ID NO: 47)GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST GPSGAP,(SEQ ID NO: 51) GAPGGGSPAPAPTPAPAPTPAPAGGGPSGAP, (SEQ ID NO: 55)GAPGGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGGGAP, and (SEQ ID NO: 71)GGGGAGGGGAGGGGAGGGGAGGGPS.

Exemplary lysosomal storage diseases contemplated by the methods hereininclude those set out in Table 1. It is contemplated that the lysosomalstorage disease is treated using a targeted therapeutic fusion proteincomprising the enzyme deficient in the lysosomal storage disease, alsodisclosed in Table 1.

In various embodiments, the invention provides a method for treatingMucopolysaccharidosis Type IIIB (Sanfilippo B Syndrome) in a subjectcomprising administering to the subject a therapeutically effectiveamount of a pharmaceutical composition comprising a fusion proteincomprising an amino acid sequence at least 85% identical to a humanα-N-acetylglucosaminidase (Naglu) protein (SEQ ID NO: 1), a peptide taghaving an amino acid sequence at least 70% identical to amino acids 8-67of mature human IGF-II and a spacer peptide located between the Nagluamino acid sequence and the IGF-II peptide tag. In various embodiments,the spacer comprises the amino acid sequence GAP (SEQ ID NO: 9), GPS(SEQ ID NO: 10), or GGS (SEQ ID NO: 11).

In various embodiments, the spacer sequence comprises amino acidsGly-Pro-Ser (GPS) (SEQ ID NO: 10) between the amino acids of maturehuman IGF-II and the amino acids of human Naglu.

In various embodiments, the spacer peptide comprises one or more GGGGS(SEQ ID NO: 12) or GGGS (SEQ ID NO: 16) amino acid sequences. In variousembodiments, the spacer peptide comprises one or more GGGPS (SEQ ID NO:14) or GGGSP (SEQ ID NO: 15) amino acid sequences. In variousembodiments, the spacer peptide comprises one or more AAAAS (SEQ ID NO:17) or AAAS (SEQ ID NO: 18) amino acid sequences. In variousembodiments, the spacer peptide comprises one or more PAPA (SEQ ID NO:19) or TPAPA (SEQ ID NO: 20) amino acid sequences. In variousembodiments, the spacer peptide comprises one or more AAAKE (SEQ ID NO:21) amino acid sequences. In various embodiments, the spacer peptidecomprises one or more GGGGA (SEQ ID NO: 60) amino acid sequences.

In various embodiments, the spacer peptide comprises an amino acidsequence selected from the group consisting of:

(SEQ ID NOs: 12, 56, 58, 91-94) (GGGGS)n, (SEQ ID NOs: 36, 95-100)(GGGGS)n-GGGPS, (SEQ ID NOs: 101-107) GAP-(GGGGS)n-GGGPS,(SEQ ID NOs: 37, 108-113) GAP-(GGGGS)n-GGGPS-GAP, (SEQ ID NOs: 114-162)GAP-(GGGGS)n-GGGPS-(GGGGS)n-GAP, (SEQ ID NOs: 163-169)GAP-GGGPS-(GGGGS)n-GAP, (SEQ ID NOs: 170-218)GAP-(GGGGS)n-AAAAS-GGGPS-(GGGGS)n-AAAA-GAP, (SEQ ID NOs: 219-267)GAP-(GGGGS)n-PAPAP-(Xaa)n-GAP, (SEQ ID NOs: 268-316)GAP-(GGGGS)n-PAPAPT-(Xaa)n-GAP, (SEQ ID NOs: 544-551)GAP-(GGGGS)n-(Xaa)n-PAPAP-(Xaa)n-(AAAKE)n-(Xaa)n- (GGGGS)n-GAP,(SEQ ID NOs: 60, 79, 81, 317-320) (GGGGA)n, (SEQ ID NOs: 321-326)(GGGGA)n-GGGPS, (SEQ ID NOs: 327-333) GAP-(GGGGA)n-GGGPS,(SEQ ID NOs: 334-340) GAP-(GGGGA)n-GGGPS-GAP, (SEQ ID NOs: 341-389)GAP-(GGGGA)n-GGGPS-(GGGGA)n-GAP, (SEQ ID NOs: 390-396)GAP-GGGPS-(GGGGA)n-GAP, (SEQ ID NOs: 397-445)GAP-(GGGGA)n-AAAAS-GGGPS-(GGGGA)n-AAAA-GAP, (SEQ ID NOs: 446-494)GAP-(GGGGA)n-PAPAP-(Xaa)n-GAP, (SEQ ID NOs: 495-543)GAP-(GGGGA)n-PAPAPT-(Xaa)n-GAP, (SEQ ID NOs: 552-559)GAP-(GGGGA)n-(Xaa)n-PAPAP-(Xaa)n-(AAAKE)n-(Xaa)n- (GGGGA)n-GAP;wherein n is 1 to 7, optionally n is 1 to 4.

In various embodiments, the invention provides a method for reducingglycosaminoglycan (GAG) levels in vivo comprising administering to asubject suffering from Mucopolysaccharidosis Type IIIB (Sanfilippo BSyndrome) an effective amount of a fusion protein comprising i) an aminoacid sequence at least 85% identical to a humanα-N-acetylglucosaminidase (Naglu) protein (SEQ ID NO: 1), ii) a peptidetag having an amino acid sequence at least 70% identical to amino acids8-67 of mature human IGF-II, and iii) a spacer peptide located betweenthe Naglu amino acid sequence and the IGF-II peptide tag.

In various embodiments, the spacer sequence comprises one or more copiesof amino acids Gly-Ala-Pro (GAP) (SEQ ID NO: 9) between the amino acidsof mature human IGF-II and the amino acids of human Naglu.

In various embodiments, the spacer peptide is selected from the groupconsisting of

(SEQ ID NO: 22) EFGGGGSTR, (SEQ ID NO: 9) GAP, (SEQ ID NO: 12) GGGGS,(SEQ ID NO: 23) GPSGSPG, (SEQ ID NO: 24) GPSGSPGT, (SEQ ID NO: 25)GPSGSPGH, (SEQ ID NO: 26) GGGGSGGGGSGGGGSGGGGSGGGPST, (SEQ ID NO: 27)GGGGSGGGGSGGGGSGGGGSGGGPSH, (SEQ ID NO: 28)GGGGSGGGGSGGGGSGGGGSGGGPSGGGGSGGGPS, (SEQ ID NO: 29)GAPGGGGSGGGGSGGGGSGGGGSGGGPSGGGGSGGGPSGAP, (SEQ ID NO: 30)GGGGSGGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGGSGGGGSGGGP S, (SEQ ID NO: 31)GAPGGGGSGGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGGSGGGGSGG GPSGAP,(SEQ ID NO: 32) GGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGPS, (SEQ ID NO: 33)GAPGGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGPSGAP, (SEQ ID NO: 34)GGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGPSGGGGSGGGGSGGGP S, (SEQ ID NO: 35)GAPGGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGPSGGGGSGGGGSGG GPSGAP,(SEQ ID NO: 36) GGGGSGGGGSGGGGSGGGGSGGGPS, (SEQ ID NO: 37)GAPGGGGSGGGGSGGGGSGGGGSGGGPSGAP, (SEQ ID NO: 38)GGGGSGGGGSAAAASGGGGSGGGPS, (SEQ ID NO: 39)GAPGGGGSGGGGSAAAASGGGGSGGGPSGAP, (SEQ ID NO: 40)GGGGSGGGGSAAAASGGGGSGGGGSAAAASGGGGSGGGGSAAAASGGGP S, (SEQ ID NO: 41)GAPGGGGSGGGGSAAAASGGGGSGGGGSAAAASGGGGSGGGGSAAAASGG GPSGAP,(SEQ ID NO: 42) GGGGSGGGGSAAAASGGGPSGGGGSAAAASGGGPSGGGGSAAAASGGGP S,(SEQ ID NO: 43) GAPGGGGSGGGGSAAAASGGGPSGGGGSAAAASGGGPSGGGGSAAAASGGGPSGAP, (SEQ ID NO: 44) GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPGPS,(SEQ ID NO: 45) GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPGPS GAP,(SEQ ID NO: 46) GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTGP S,(SEQ ID NO: 47) GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTGPSGAP, (SEQ ID NO: 48) GGGSPAPTPTPAPTPAPTPAGGGPS, (SEQ ID NO: 49)GAPGGGSPAPTPTPAPTPAPTPAGGGPSGAP, (SEQ ID NO: 50)GGGSPAPAPTPAPAPTPAPAGGGPS, (SEQ ID NO: 51)GAPGGGSPAPAPTPAPAPTPAPAGGGPSGAP, (SEQ ID NO: 52)GGGSAEAAAKEAAAKEAAAKAGGPS, (SEQ ID NO: 53)GAPGGGSAEAAAKEAAAKEAAAKAGGPSGAP, (SEQ ID NO: 54)GGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGG, (SEQ ID NO: 55)GAPGGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGGGAP, (SEQ ID NO: 56)GGGGSGGGGSGGGGS, (SEQ ID NO: 57) GAPGGGGSGGGGSGGGGSGAP, (SEQ ID NO: 58)GGGGSGGGGSGGGGSGGGGS, (SEQ ID NO: 59) GAPGGGGSGGGGSGGGGSGGGGSGAP,(SEQ ID NO: 60) GGGGA, (SEQ ID NO: 61) GGGGAGGGGAGGGGAGGGGAGGGPST,(SEQ ID NO: 62) GGGGAGGGGAGGGGAGGGGAGGGPSH, (SEQ ID NO: 63)GGGGAGGGGAGGGGAGGGGAGGGPSGGGGAGGGPS, (SEQ ID NO: 64)GAPGGGGAGGGGAGGGGAGGGGAGGGPSGGGGAGGGPSGAP, (SEQ ID NO: 65)GGGGAGGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGGAGGGGAGGGP S, (SEQ ID NO: 66)GAPGGGGAGGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGGAGGGGAGG GPSGAP,(SEQ ID NO: 67) GGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGPS, (SEQ ID NO: 68)GAPGGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGPSGAP, (SEQ ID NO: 69)GGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGPSGGGGAGGGGAGGGP S, (SEQ ID NO: 70)GAPGGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGPSGGGGAGGGGAGG GPSGAP,(SEQ ID NO: 71) GGGGAGGGGAGGGGAGGGGAGGGPS, (SEQ ID NO: 72)GAPGGGGAGGGGAGGGGAGGGGAGGGPSGAP, (SEQ ID NO: 73)GGGGAGGGGAAAAASGGGGAGGGPS, (SEQ ID NO: 74)GAPGGGGAGGGGAAAAASGGGGAGGGPSGAP, (SEQ ID NO: 75)GGGGAGGGGAAAAASGGGGAGGGGAAAAASGGGGAGGGGAAAAASGGGP S, (SEQ ID NO: 76)GAPGGGGAGGGGAAAAASGGGGAGGGGAAAAASGGGGAGGGGAAAAASGG GPSGAP,(SEQ ID NO: 77) GGGGAGGGGAAAAASGGGPSGGGGAAAAASGGGPSGGGGAAAAASGGGP S,(SEQ ID NO: 78) GAPGGGGAGGGGAAAAASGGGPSGGGGAAAAASGGGPSGGGGAAAAASGGGPSGAP, (SEQ ID NO: 79) GGGGAGGGGAGGGGA, (SEQ ID NO: 80)GAPGGGGAGGGGAGGGGAGAP, (SEQ ID NO: 81) GGGGAGGGGAGGGGAGGGGA,(SEQ ID NO: 82) GAPGGGGAGGGGAGGGGAGGGGAGAP, (SEQ ID NO: 83)GGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGPS [or (GGGGA)8GGGPS],(SEQ ID NO: 84) GGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGPSH[or (GGGGA)8GGGPSH], (SEQ ID NO: 85)GGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGPS [or (GGGGA)9GGGPS],(SEQ ID NO: 86) GGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGPSH [or (GGGGA)9GGGPSH,] (SEQ ID NO: 87) GGGGPAPGPGPAPGPAPGPAGGGPS,(SEQ ID NO: 88) GAPGGGGPAPGPGPAPGPAPGPAGGGPGGAP, (SEQ ID NO: 89)GGGGPAPAPGPAPAPGPAPAGGGPS, and (SEQ ID NO: 90)GAPGGGGPAPAPGPAPAPGPAPAGGGPGGAP.

In various embodiments, the spacer peptide is selected from the groupconsisting of

(SEQ ID NO: 36) GGGGSGGGGSGGGGSGGGGSGGGPS, (SEQ ID NO: 44)GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPGPS, (SEQ ID NO: 45)GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPGPS GAP, (SEQ ID NO: 46)GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTGP S, (SEQ ID NO: 47)GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST GPSGAP,(SEQ ID NO: 48) GGGSPAPTPTPAPTPAPTPAGGGPS, (SEQ ID NO: 49)GAPGGGSPAPTPTPAPTPAPTPAGGGPSGAP, (SEQ ID NO: 50)GGGSPAPAPTPAPAPTPAPAGGGPS, (SEQ ID NO: 51)GAPGGGSPAPAPTPAPAPTPAPAGGGPSGAP, (SEQ ID NO: 52)GGGSAEAAAKEAAAKEAAAKAGGPS, (SEQ ID NO: 53)GAPGGGSAEAAAKEAAAKEAAAKAGGPSGAP, (SEQ ID NO: 54)GGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGG, (SEQ ID NO: 55)GAPGGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGGGAP, and (SEQ ID NO: 71)GGGGAGGGGAGGGGAGGGGAGGGPS.

In various embodiments, the spacer peptide is selected from the groupconsisting of

(SEQ ID NO: 36) GGGGSGGGGSGGGGSGGGGSGGGPS, (SEQ ID NO: 47)GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST GPSGAP,(SEQ ID NO: 51) GAPGGGSPAPAPTPAPAPTPAPAGGGPSGAP, (SEQ ID NO: 55)GAPGGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGGGAP, and (SEQ ID NO: 71)GGGGAGGGGAGGGGAGGGGAGGGPS.

In various embodiments, the lysosomal targeting domain or IGF-II peptidetag comprises amino acids 8-67 of mature human IGF-II (SEQ ID NO: 2, 4).In various embodiments, the IGF-II peptide tag comprises a mutation atresidue Arg37. In various embodiments, the mutation is a substitution ofalanine for arginine. In various embodiments, the lysosomal targetingdomain or IGF-II peptide tag comprises IGF2 Δ8-67 R37A.

In various embodiments, the fusion protein comprises amino acids 1-743of human Naglu (SEQ ID NO: 1, 3). In various embodiments, the fusionprotein comprises amino acids 24-743 of human Naglu.

In various embodiments, the effective amount of fusion protein is in therange of about 0.1-1 mg/kg, about 1-5 mg/kg, about 2.5-20 mg/kg, about5-20 mg/kg, about 10-50 mg/kg, or 20-100 mg/kg of body weight of thesubject. In various embodiments, the effective amount of fusion proteinis about 2.5-20 mg per kilogram of body weight of the subject.

In various embodiments, the fusion protein is administeredintrathecally, intravenously, intramuscularly, parenterally,transdermally, or transmucosally. In various embodiments, the fusionprotein is administered intrathecally. In various embodiments, theintrathecal administration optionally further comprises administeringthe fusion protein intravenously.

In various embodiments, intrathecal administration comprises introducingthe fusion protein into a cerebral ventricle, lumbar area, or cisternamagna.

In various embodiments, the fusion protein is administered bimonthly,monthly, triweekly, biweekly, weekly, daily, or at variable intervals.

In various embodiments, the treatment results in reducingglycosaminoglycan (GAG) levels in a brain tissue. It is furthercontemplated that the treatment results in reducing lysosomal storagegranules in a brain tissue.

Also contemplated are compositions comprising the targeted therapeuticfusion proteins as described herein for use in treating lysosomalstorage diseases. Exemplary lysosomal storage diseases include those setout in Table 1.

Other features, objects, and advantages of the present invention areapparent in the detailed description that follows. It should beunderstood, however, that the detailed description, while indicatingembodiments of the present invention, is given by way of illustrationonly, not limitation. Various changes and modifications within the scopeof the invention will become apparent to those skilled in the art fromthe detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are for illustration purposes only, not for limitation.

FIG. 1 depicts the amino acid sequences of a portion of an exemplarytherapeutic fusion protein comprising (A) Naglu and (B) an IGF-IIpeptide comprising residues 8-67 of IGF-II and having an amino acidsubstitution at residue 37, R37A (Arg37Ala).

FIG. 2 depicts the nucleotide sequences of a portion of an exemplarytherapeutic fusion protein comprising (A) Naglu and (B) an IGF-IIpeptide comprising residues 8-67 of IGF-II and having an amino acidsubstitution at residue 37, R37A (Arg37Ala).

FIG. 3 discloses exemplary spacer sequences contemplated for use in thetherapeutic fusion protein.

DEFINITIONS

Amelioration: As used herein, the term “amelioration” is meant theprevention, reduction or palliation of a state, or improvement of thestate of a subject. Amelioration includes, but does not require completerecovery or complete prevention of a disease condition. In someembodiments, amelioration includes reduction of accumulated materialsinside lysosomes of relevant diseases tissues.

Furin-resistant IGF-II mutein: As used herein, the term “furin-resistantIGF-II mutein” refers to an IGF-II-based peptide containing an alteredamino acid sequence that abolishes at least one native furin proteasecleavage site or changes a sequence close or adjacent to a native furinprotease cleavage site such that the furin cleavage is prevented,inhibited, reduced, or slowed down as compared to a wild-type humanIGF-II peptide. As used herein, a furin-resistant IGF-II mutein is alsoreferred to as an IGF-II mutein that is resistant to furin.

Furin protease cleavage site: As used herein, the term “furin proteasecleavage site” (also referred to as “furin cleavage site” or “furincleavage sequence”) refers to the amino acid sequence of a peptide orprotein that serves as a recognition sequence for enzymatic proteasecleavage by furin or furin-like proteases. Typically, a furin proteasecleavage site has a consensus sequence Arg-X-X-Arg (SEQ ID NO: 6), X isany amino acid. The cleavage site is positioned after thecarboxy-terminal arginine (Arg) residue in the sequence. In someembodiments, a furin cleavage site may have a consensus sequenceLys/Arg-X-X-X-Lys/Arg-Arg (SEQ ID NO: 7), X is any amino acid. Thecleavage site is positioned after the carboxy-terminal arginine (Arg)residue in the sequence.

Furin: As used herein, the term “furin” refers to any protease that canrecognize and cleave the furin protease cleavage site as defined herein,including furin or furin-like protease. Furin is also known as pairedbasic amino acid cleaving enzyme (PACE). Furin belongs to thesubtilisin-like proprotein convertase family. The gene encoding furinwas known as FUR (FES Upstream Region).

Furin-deficient cells: As used herein, the term “furin-deficient cells”refers to any cells whose furin protease activity is inhibited, reducedor eliminated. Furin-deficient cells include both mammalian andnon-mammalian cells that do not produce furin or produce reduced amountof furin or defective furin protease.

Glycosylation Independent Lysosomal Targeting: As used herein, the term“glycosylation independent lysosomal targeting” (also referred to as“GILT”) refer to lysosomal targeting that ismannose-6-phosphate-independent.

Human Alpha-N-acetylglucosaminidase: As used herein, the term “humanalpha-N-acetylglucosaminidase” (also referred to as “Naglu”) refers toprecursor (i.e., containing the native Naglu signal peptide sequence) orprocessed (i.e., lacking the native Naglu signal peptide sequence)wild-type form of human alpha-N-acetylglucosaminidase, or a functionalfragment or variant thereof, that is capable of reducingglycosaminoglycan (GAG) levels in mammalian lysosomes or that can rescueor ameliorate one or more MPS IIIB (Sanfilippo B Syndrome) symptoms. Asused herein, the term “functional” as it relates to Naglu refers to aNaglu enzyme that is capable of being taken up by mammalian lysosomesand having sufficient enzymatic activity to reduce storage material,i.e., glycosaminoglycan (GAG), in the mammalian lysosome.

IGF-II mutein: As used herein, the term “IGF-II mutein” refers to anIGF-II-based peptide containing an altered amino acid sequence. As usedherein, the term “furin-resistant IGF-II mutein” refers to anIGF-II-based peptide containing an altered amino acid sequence thatabolishes at least one native furin protease cleavage site or changes asequence close or adjacent to a native furin protease cleavage site suchthat the furin cleavage is prevented, inhibited, reduced, or slowed downas compared to a wild-type human IGF-II peptide. As used herein, afurin-resistant IGF-II mutein is also referred to as an IGF-II muteinthat is resistant to furin.

Improve, increase, or reduce: As used herein, the terms “improve,”“increase” or “reduce,” or grammatical equivalents, indicate values thatare relative to a baseline measurement, such as a measurement in thesame individual prior to initiation of the treatment described herein,or a measurement in a control individual (or multiple controlindividuals) in the absence of the treatment described herein. A“control individual” is an individual afflicted with the same form oflysosomal storage disease (e.g., MPS IIIB (Sanfilippo B Syndrome)) asthe individual being treated, who is about the same age as theindividual being treated (to ensure that the stages of the disease inthe treated individual and the control individual(s) are comparable).

Individual, subject, patient: As used herein, the terms “subject,”“individual” or “patient” refer to a human or a non-human mammaliansubject. The individual (also referred to as “patient” or “subject”)being treated is an individual (fetus, infant, child, adolescent, oradult human) suffering from a lysosomal storage disease, for example,MPS IIIB (Sanfilippo B Syndrome) (i.e., either infantile-, juvenile-, oradult-onset or severe/classical type or attenuated type MPS IIIB(Sanfilippo B Syndrome)) or having the potential to develop a lysosomalstorage disease (e.g., MPS IIIB (Sanfilippo B Syndrome)).

Lysosomal storage diseases: As used herein, “lysosomal storage diseases”refer to a group of genetic disorders that result from deficiency in atleast one of the enzymes (e.g., acid hydrolases) that are required tobreak macromolecules down to peptides, amino acids, monosaccharides,nucleic acids and fatty acids in lysosomes. As a result, individualssuffering from lysosomal storage diseases have accumulated materials inlysosomes. Exemplary lysosomal storage diseases are listed in Table 1.

Lysosomal enzyme: As used herein, the term “lysosomal enzyme” refers toany enzyme that is capable of reducing accumulated materials inmammalian lysosomes or that can rescue or ameliorate one or morelysosomal storage disease symptoms. Lysosomal enzymes suitable for theinvention include both wild-type or modified lysosomal enzymes and canbe produced using recombinant and synthetic methods or purified fromnature sources. Exemplary lysosomal enzymes are listed in Table 1.

Spacer: As used herein, the term “spacer” (also referred to as “linker”)refers to a peptide sequence between two protein moieties in a fusionprotein. A spacer is generally designed to be flexible or to interpose astructure, such as an alpha-helix, between the two protein moieties. Aspacer can be relatively short, such for example, the sequenceGly-Ala-Pro (GAP) (SEQ ID NO: 9), Gly-Gly-Gly-Gly-Ser (GGGGS) (SEQ IDNO: 12), Gly-Gly-Gly-Gly-Ala (GGGGA) (SEQ ID NO: 60) orGly-Gly-Gly-Gly-Gly-Pro (GGGGGP) (SEQ ID NO: 13), or can be longer, suchas, for example, 10-25 amino acids in length, 25-50 amino acids inlength or 35-55 amino acids in length. Exemplary spacer sequences aredisclosed in greater detail in the Detailed Description.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” or “effective amount” refers to anamount of a targeted therapeutic fusion protein which confers atherapeutic effect on the treated subject, at a reasonable benefit/riskratio applicable to any medical treatment. The therapeutic effect may beobjective (i.e., measurable by some test or marker) or subjective (i.e.,subject gives an indication of or feels an effect). In particular, the“therapeutically effective amount” refers to an amount of a therapeuticfusion protein or composition effective to treat, ameliorate, or preventa desired disease or condition, or to exhibit a detectable therapeuticor preventative effect, such as by ameliorating symptoms associated withthe disease, preventing or delaying the onset of the disease, and/oralso lessening the severity or frequency of symptoms of the disease. Atherapeutically effective amount is commonly administered in a dosingregimen that may comprise multiple unit doses. For any particulartherapeutic fusion protein, a therapeutically effective amount (and/oran appropriate unit dose within an effective dosing regimen) may vary,for example, depending on route of administration, on combination withother pharmaceutical agents. Also, the specific therapeuticallyeffective amount (and/or unit dose) for any particular patient maydepend upon a variety of factors including the disorder being treatedand the severity of the disorder; the activity of the specificpharmaceutical agent employed; the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and/or rate of excretion ormetabolism of the specific fusion protein employed; the duration of thetreatment; and like factors as is well known in the medical arts.

Treatment: As used herein, the term “treatment” (also “treat” or“treating”) refers to any administration of a therapeutic fusion proteinor pharmaceutical composition comprising said therapeutic fusion proteinthat partially or completely alleviates, ameliorates, relieves,inhibits, delays onset of, reduces severity of and/or reduces incidenceof one or more symptoms or features of a particular disease, disorder,and/or condition. Such treatment may be of a subject who does notexhibit signs of the relevant disease, disorder and/or condition and/orof a subject who exhibits only early signs of the disease, disorder,and/or condition. Alternatively or additionally, such treatment may beof a subject who exhibits one or more established signs of the relevantdisease, disorder and/or condition. For example, treatment can refer toimprovement of cardiac status (e.g., increase of end-diastolic and/orend-systolic volumes, or reduction, amelioration or prevention of theprogressive cardiomyopathy that is typically found in, e.g., Pompedisease) or of pulmonary function (e.g., increase in crying vitalcapacity over baseline capacity, and/or normalization of oxygendesaturation during crying); improvement in neurodevelopment and/ormotor skills (e.g., increase in AIMS score); reduction of storage (e.g.,glycosaminoglycan (GAG), levels in tissue of the individual affected bythe disease; or any combination of these effects. In some embodiments,treatment includes improvement of glycosaminoglycan (GAG) clearance,particularly in reduction or prevention of MPS IIIB (Sanfilippo BSyndrome)-associated neuronal symptoms.

As used in this application, the terms “about” and “approximately” areused as equivalents. Any numerals used in this application with orwithout about/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides improved methods and compositions fortargeting lysosomal enzymes based on the glycosylation-independentlysosomal targeting (GILT) technology. Among other things, the presentinvention provides IGF-II muteins that are resistant to furin and/or hasreduced or diminished binding affinity for the insulin receptor, and/orhas reduced or diminished binding affinity for the IGF-I receptor andtargeted therapeutic fusion proteins containing an IGF-II mutein of theinvention. The present invention also provides methods of making andusing the same.

Various aspects of the invention are described in detail in thefollowing sections. The use of sections is not meant to limit theinvention. Each section can apply to any aspect of the invention. Inthis application, the use of “or” means “and/or” unless statedotherwise.

Lysosomal Enzymes

A lysosomal enzyme suitable for the invention includes any enzyme thatis capable of reducing accumulated materials in mammalian lysosomes orthat can rescue or ameliorate one or more lysosomal storage diseasesymptoms. Suitable lysosomal enzymes include both wild-type or modifiedlysosomal enzymes and can be produced using recombinant or syntheticmethods or purified from natural sources. Exemplary lysosomal enzymesare listed in Table 1.

TABLE 1 Lysosomal Storage Diseases and associated enzyme defects DiseaseName Enzyme Defect Substance Stored A. Glycogenosis Disorders PompeDisease Acid-α1, 4-Glucosidase Glycogen α1-4 linked Oligosaccharides B.Glycolipidosis Disorders GM1 Gangliodsidosis β-Galactosidase GM₁Gangliosides Tay-Sachs Disease β-Hexosaminidase A GM₂ Ganglioside GM2Gangliosidosis: AB GM₂ Activator Protein GM₂ Ganglioside VariantSandhoff Disease β-Hexosaminidase A&B GM₂ Ganglioside Fabry Diseaseα-Galactosidase A Globosides Gaucher Disease GlucocerebrosidaseGlucosylceramide Metachromatic Arylsulfatase A SulphatidesLeukodystrophy Krabbe Disease Galactosylceramidase GalactocerebrosideNiemann-Pick, Types A & B Acid Sphingomyelinase SphingomyelinNiemann-Pick, Type C Cholesterol Esterification Sphingomyelin DefectNiemann-Pick, Type D Unknown Sphingomyelin Farber Disease AcidCeramidase Ceramide Wolman Disease Acid Lipase Cholesteryl Esters C.Mucopolysaccharide Disorders Hurler Syndrome (MPS IH) α-L-IduronidaseHeparan & Dermatan Sulfates Scheie Syndrome (MPS IS) α-L-IduronidaseHeparan & Dermatan Sulfates Hurler-Scheie (MPS IH/S) α-L-IduronidaseHeparan & Dermatan Sulfates Hunter Syndrome (MPS II) Iduronate SulfataseHeparan & Dermatan Sulfates Sanfilippo A (MPS IIIA) Heparan N-SulfataseHeparan Sulfate Sanfilippo B (MPS IIIB) α-N-AcetylglucosaminidaseHeparan Sulfate Sanfilippo C (MPS IIIC) Acetyl-CoA-Glucosaminide HeparanSulfate Acetyltransferase Sanfilippo D (MPS IIID) N-Acetylglucosamine-6-Heparan Sulfate Sulfatase Morquio A (MPS IVA) Galactosamine-6-SulfataseKeratan Sulfate Morquio B (MPS IVB) β-Galactosidase Keratan SulfateMaroteaux-Lamy (MPS VI) Arylsulfatase B Dermatan Sulfate Sly Syndrome(MPS VII) β-Glucuronidase D. Oligosaccharide/Glycoprotein Disordersα-Mannosidosis α-Mannosidase Mannose/Oligosaccharides β-Mannosidosisβ-Mannosidase Mannose/Oligosaccharides Fucosidosis α-L-FucosidaseFucosyl Oligosaccharides Aspartylglucosaminuria N-Aspartyl-β-Aspartylglucosamine Glucosaminidase Asparagines Sialidosis(Mucolipidosis I) α-Neuraminidase SialyloligosaccharidesGalactosialidosis Lysosomal Protective Sialyloligosaccharides (GoldbergSyndrome) Protein Deficiency Schindler Disease α-N-Acetyl-Galactosaminidase E. Lysosomal Enzyme Transport Disorders MucolipidosisII (I-Cell N-Acetylglucosamine-1- Heparan Sulfate Disease)Phosphotransferase Mucolipidosis III (Pseudo- Same as ML II HurlerPolydystrophy) F. Lysosomal Membrane Transport Disorders CystinosisCystine Transport Protein Free Cystine Salla Disease Sialic AcidTransport Protein Free Sialic Acid and Glucuronic Acid Infantile SialicAcid Storage Sialic Acid Transport Protein Free Sialic Acid and DiseaseGlucuronic Acid G. Other Batten Disease Unknown Lipofuscins (JuvenileNeuronal Ceroid Lipofuscinosis) Infantile Neuronal CeroidPalmitoyl-Protein Thioesterase Lipofuscins Lipofuscinosis Late InfantileNeuronal Tripeptidyl Peptidase I Lipofuscins Ceroid LipofuscinosisMucolipidosis IV Unknown Gangliosides & Hyaluronic Acid ProsaposinSaposins A, B, C or D

In some embodiments, a lysosomal enzyme contemplated herein includes apolypeptide sequence having 50-100%, including 50%, 55%, 60%, 65%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% and 100%, sequence identity to the naturally-occurringpolynucleotide sequence of a human enzyme shown in Table 1, while stillencoding a protein that is functional, i.e., capable of reducingaccumulated materials, e.g., glycosaminoglycan (GAG), in mammalianlysosomes or that can rescue or ameliorate one or more lysosomal storagedisease symptoms.

“Percent (%) amino acid sequence identity” with respect to the lysosomalenzyme sequences is defined as the percentage of amino acid residues ina candidate sequence that are identical with the amino acid residues inthe naturally-occurring human enzyme sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, ALIGN or Megalign(DNASTAR) software. Those skilled in the art can determine appropriateparameters for measuring alignment, including any algorithms needed toachieve maximal alignment over the full length of the sequences beingcompared. Preferably, the WU-BLAST-2 software is used to determine aminoacid sequence identity (Altschul et al., Methods in Enzymology 266,460-480 (1996). WU-BLAST-2 uses several search parameters, most of whichare set to the default values. The adjustable parameters are set withthe following values: overlap span=1, overlap fraction=0.125, worldthreshold (T)=11. HSP score (S) and HSP S2 parameters are dynamic valuesand are established by the program itself, depending upon thecomposition of the particular sequence, however, the minimum values maybe adjusted and are set as indicated above.

Alpha-N-Acetylglucosaminidase

Alpha-N-acetylglucosaminidase, Naglu, is produced as a precursormolecule that is processed to a mature form. This process generallyoccurs by removing the 23 amino acid signal peptide as the proteinenters the endoplasmic reticulum. Typically, the precursor form is alsoreferred to as full-length precursor or full-length Naglu protein, whichcontains 743 amino acids (SEQ ID NO: 1). The N-terminal 23 amino acidsare cleaved as the precursor protein enters the endoplasmic reticulum,resulting in a processed or mature form. Thus, it is contemplated thatthe N-terminal 23 amino acids are generally not required for the Nagluprotein activity. The amino acid sequences of the mature form andfull-length precursor form of a typical wild-type or naturally-occurringhuman Naglu protein are shown in FIG. 1 and set out in SEQ ID NO: 1. Thenucleotide sequence of the coding region of human Naglu is set out inSEQ ID NO: 3. The mRNA sequence of human Naglu is described in GenbankAccession number NM_000263. In various embodiments, the Naglu is humanNaglu, with (amino acids 1-743) or without (amino acids 24-743) signalsequence.

U.S. Pat. No. 6,255,096 describes that the molecular weight of purifiedhuman alpha-N-acetylglucosaminidase (i.e. 82 kDa and 77 kDa) andrecombinant mammalian alpha-N-acetylglucosaminidase produced in CHOcells (i.e. 89 kDa and 79 kDa) are greater than the deduced molecularweight of the Naglu polypeptide (i.e. 70 kDa), suggesting that thepurified and recombinant polypeptide are post-translationally modified.See also Weber et al., Hum Mol Genet 5:771-777, 1996.

Mucopolysaccharidosis III B (Sanfilippo B Syndrome)

One exemplary lysosomal storage disease is Mucopolysaccharidosis III B(MPS IIIB) disease, also known as Sanfilippo Type B Syndrome. MPS IIIB,Sanfilippo B Syndrome, is a rare autosomal recessive genetic disorderthat is characterized by a deficiency of the enzymealpha-N-acetyl-glucosaminidase (Naglu). In the absence of this enzyme,glycosaminoglycans (GAG), for example the GAG heparan sulfate, andpartially degraded GAG molecules cannot be cleared from the body andaccumulate in lysosomes of various tissues, resulting in progressivewidespread somatic dysfunction (Kakkis et al., N Engl J Med.344(3):182-8, 2001). It has been shown that GAGs accumulate in lysosomesof neurons and glial cells, with lesser accumulation outside the brain.

Four distinct forms of MPS III, designated MPS IIIA, B, C, and D, havebeen identified. Each represents a deficiency in one of four enzymesinvolved in the degradation of the GAG heparan sulfate (Table 1). Allforms include varying degrees of the same clinical symptoms, includingcoarse facial features, hepatosplenomegaly, corneal clouding andskeletal deformities. Most notably, however, is the severe andprogressive loss of cognitive ability, which is tied not only to theaccumulation of heparan sulfate in neurons, but also the subsequentelevation of the gangliosides GM2, GM3 and GD2 caused by primary GAGaccumulation (Walkley et al., Ann N Y Acad Sci. 845:188-99, 1998).

A characteristic clinical feature of Sanfilippo B Syndrome is centralnervous system (CNS) degeneration, which results in loss of, or failureto attain, major developmental milestones. The progressive cognitivedecline culminates in dementia and premature mortality. The diseasetypically manifests itself in young children, and the lifespan of anaffected individual generally does not extend beyond late teens to earlytwenties.

MPS III diseases all have similar symptoms that typically manifest inyoung children. Affected infants are apparently normal, although somemild facial dysmorphism may be noticeable. The stiff joints, hirsutenessand coarse hair typical of other mucopolysaccharidoses are usually notpresent until late in the disease. After an initial symptom-freeinterval, patients usually present with a slowing of development and/orbehavioral problems, followed by progressive intellectual declineresulting in severe dementia and progressive motor disease. Acquisitionof speech is often slow and incomplete. The disease progresses toincreasing behavioral disturbance including temper tantrums,hyperactivity, destructiveness, aggressive behavior, pica and sleepdisturbance. As affected children have normal muscle strength andmobility, the behavioral disturbances are very difficult to manage. Inthe final phase of the illness, children become increasingly immobileand unresponsive, often require wheelchairs, and develop swallowingdifficulties and seizures. The life-span of an affected child does notusually extend beyond late teens to early twenties.

An alpha-N-acetylglucosaminidase enzyme suitable for treating MPS IIIB(Sanfilippo B Syndrome) includes a wild-type humanalpha-N-acetylglucosaminidase (SEQ ID NO: 1 or 3), or a functionalfragment or sequence variant thereof which retains the ability to betaken up into mammalian lysosomes and to hydrolyze alpha, 1,4 linkagesat the terminal N-acetyl-D-glucosamine residue in linearoligosaccharides.

Efficacy of treatment of MPS IIIB (Sanfilippo B Syndrome) usingrecombinant targeted therapeutic fusion proteins as described herein canbe measured using techniques known in the art, as well as by analysis oflysosomal and neuronal biomarkers. Initial experiments are conducted onNaglu knock-out animals (see Li et al., Proc Natl Acad Sci USA96:14505-510, 1999). Naglu knockouts present with large amounts ofheparan sulfate in the liver and kidney and elevation of gangliosides inbrain.

Assays include analysis of the activity of and biodistribution of theexogenous enzyme, reduction of GAG storage in the lysosomes,particularly in brain cells, and activation of astrocytes and microglia.Levels of various lysosomal or neuronal biomarkers include, but are notlimited to, Lysosomal-associated membrane protein 1 (LAMP1), glypican,gangliosides, cholesterol, Subunit c of Mitochondrial ATP Synthase(SCMAS), ubiquitin, P-GSK3b, beta amyloid and P-tau. Survival andbehavioral analysis is also performed using techniques known in thefield.

Experiments have shown that Subunit c of Mitochondrial ATP Synthase(SCMAS) protein accumulates in the lysosomes of MPS IIIB animals(Ryazantsev et al., Mol Genet Metab. 90(4): 393-401, 2007). LAMP-1 andGM130 have also been shown to be elevated in MPS IIIB animals (Vitry etal., Am J Pathol. 177(6):2984-99, 2010).

In various embodiments, treatment of a lysosomal storage disease refersto decreased lysosomal storage (e.g., of GAG) in various tissues. Invarious embodiments, treatment refers to decreased lysosomal storage inbrain target tissues, spinal cord neurons, and/or peripheral targettissues. In certain embodiments, lysosomal storage is decreased by about5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 100% or more as compared to a control. Invarious embodiments, lysosomal storage is decreased by at least 1-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-foldor more as compared to a control.

In various embodiments, treatment refers to increased enzyme activity invarious tissues. In various embodiments, treatment refers to increasedenzyme activity in brain target tissues, spinal cord neurons and/orperipheral target tissues. In various embodiments, enzyme activity isincreased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%,600%, 700%, 800%, 900% 1000% or more as compared to a control. Invarious embodiments, enzyme activity is increased by at least 1-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-foldor more as compared to a control. In various embodiments, increasedenzymatic activity is at least approximately 10 nmol/hr/mg, 20nmol/hr/mg, 40 nmol/hr/mg, 50 nmol/hr/mg, 60 nmol/hr/mg, 70 nmol/hr/mg,80 nmol/hr/mg, 90 nmol/hr/mg, 100 nmol/hr/mg, 150 nmol/hr/mg, 200nmol/hr/mg, 250 nmol/hr/mg, 300 nmol/hr/mg, 350 nmol/hr/mg, 400nmol/hr/mg, 450 nmol/hr/mg, 500 nmol/hr/mg, 550 nmol/hr/mg, 600nmol/hr/mg or more. In various embodiments, the lysosomal enzyme isNaglu.

Enzyme Replacement Therapy

Enzyme replacement therapy (ERT) is a therapeutic strategy to correct anenzyme deficiency by infusing the missing enzyme into the bloodstream.As the blood perfuses patient tissues, enzyme is taken up by cells andtransported to the lysosome, where the enzyme acts to eliminate materialthat has accumulated in the lysosomes due to the enzyme deficiency. Forlysosomal enzyme replacement therapy to be effective, the therapeuticenzyme must be delivered to lysosomes in the appropriate cells intissues where the storage defect is manifest. Conventional lysosomalenzyme replacement therapeutics are delivered using carbohydratesnaturally attached to the protein to engage specific receptors on thesurface of the target cells. One receptor, the cation-independent M6Preceptor (CI-MPR), is particularly useful for targeting replacementlysosomal enzymes because the CI-MPR is present on the surface of mostcell types.

The terms “cation-independent mannose-6-phosphate receptor (CI-MPR),”“M6P/IGF-II receptor,” “CI-MPR/IGF-II receptor,” “IGF-II receptor” or“IGF2 Receptor,” or abbreviations thereof, are used interchangeablyherein, referring to the cellular receptor which binds both M6P andIGF-II.

Combination Therapy to Tolerize Subject to Enzyme Replacement Therapy

It has been found that during administration of agents such asrecombinant proteins and other therapeutic agents, a subject can mountan immune response against these agents, leading to the production ofantibodies that bind and interfere with the therapeutic activity as wellas cause acute or chronic immunologic reactions. This problem is mostsignificant for protein therapeutics because proteins are complexantigens and in many cases, the subject is immunologically naive to theantigens. Thus, in certain aspects of the present invention, it may beuseful to render the subject receiving the therapeutic enzyme tolerantto the enzyme replacement therapy. In this context, the enzymereplacement therapy may be given to the subject as a combination therapywith a tolerizing regimen.

U.S. Pat. No. 7,485,314 (incorporated herein by reference) disclosestreatment of lysosomal storage disorders using immune toleranceinduction. Briefly, use of such a tolerization regimen may be useful toprevent the subject mounting an immune response to the enzymereplacement therapy and thereby decreasing or otherwise renderingineffective the potential beneficial effects of the enzyme replacementtherapy.

In one method, the invention contemplates reducing or preventing aclinically significant antigen-specific immune response to recombinanttherapeutic fusion protein, for example, comprising Naglu, used to treata lysosomal storage disorder, for example mucopolysaccharidosis IIIB(MPS IIIB or Sanfilippo B Syndrome), where the fusion protein isadministered intrathecally. The method employs an initial 30-60 dayregimen of a T-cell immunosuppressive agent such as cyclosporin A (CsA)and an antiproliferative agent, such as, azathioprine (Aza), combinedwith weekly intrathecal infusions of low doses of the enzyme, e.g.,Naglu. The typical strong IgG response to weekly infusions of enzymebecomes greatly reduced or prevented using a 60 day regimen ofimmunosuppressive drugs, cyclosporin A (CsA) and azathioprine (Aza),combined with weekly intrathecal or intravenous infusions of low dosesof fusion protein comprising enzyme. Using such tolerization regimens,it will be possible to render the subject tolerant to higher therapeuticdoses of therapeutic fusion protein for up to 6 months without anincrease in antibody titer against Naglu, or indeed any other enzymethat could be used for enzyme replacement of a lysosomal storagedisease. Such tolerization regimens have been described in U.S. Pat. No.7,485,314.

Glycasylation Independent Lysosomal Targeting

A Glycosylation Independent Lysosomal Targeting (GILT) technology wasdeveloped to target therapeutic enzymes to lysosomes. Specifically, theGILT technology uses a peptide tag instead of M6P to engage the CI-MPRfor lysosomal targeting. Typically, a GILT tag is a protein, peptide, orother moiety that binds the CI-MPR in a mannose-6-phosphate-independentmanner. Advantageously, this technology mimics the normal biologicalmechanism for uptake of lysosomal enzymes, yet does so in a mannerindependent of mannose-6-phosphate.

A preferred GILT tag is derived from human insulin-like growth factor II(IGF-II). Human IGF-II is a high affinity ligand for the CI-MPR, whichis also referred to as IGF-II receptor. Binding of GILT-taggedtherapeutic enzymes to the M6P/IGF-II receptor targets the protein tothe lysosome via the endocytic pathway. This method has numerousadvantages over methods involving glycosylation including simplicity andcost effectiveness, because once the protein is isolated, no furthermodifications need be made.

Detailed description of the GILT technology and GILT tag can be found inU.S. Publication Nos. 20030082176, 20040006008, 20040005309, and20050281805, the teachings of all of which are hereby incorporated byreferences in their entireties.

Furin-Resistant GILT Tag

During the course of development of GILT-tagged lysosomal enzymes fortreating lysosomal storage disease, it has become apparent that theIGF-II derived GILT tag may be subjected to proteolytic cleavage byfurin during production in mammalian cells (see the examples section).Furin protease typically recognizes and cleaves a cleavage site having aconsensus sequence Arg-X-X-Arg (SEQ ID NO: 6), X is any amino acid. Thecleavage site is positioned after the carboxy-terminal arginine (Arg)residue in the sequence. In some embodiments, a furin cleavage site hasa consensus sequence Lys/Arg-X-X-X-Lys/Arg-Arg (SEQ ID NO: 7), X is anyamino acid. The cleavage site is positioned after the carboxy-terminalarginine (Arg) residue in the sequence. As used herein, the term “furin”refers to any protease that can recognize and cleave the furin proteasecleavage site as defined herein, including furin or furin-like protease.Furin is also known as paired basic amino acid cleaving enzyme (PACE).Furin belongs to the subtilisin-like proprotein convertase family thatincludes PC3, a protease responsible for maturation of proinsulin inpancreatic islet cells. The gene encoding furin was known as FUR (FESUpstream Region).

The mature human IGF-II peptide sequence is shown below.

(SEQ ID NO: 5) AYRPSETLCGGELVDTLQFVCGDRGFYFSRPAS RVSR ↑ RSR ↑ GIVEECCFRSCDLALLETYC ATPAKSE

As can be seen, the mature human IGF-II contains two potentialoverlapping furin cleavage sites between residues 34-40 (bolded andunderlined). Arrows are inserted at two potential furin cleavagepositions.

Modified GILT tags that are resistant to cleavage by furin and stillretain ability to bind to the CI-MPR in amannose-6-phosphate-independent manner are disclosed in US 20110223147.Specifically, furin-resistant GILT tags can be designed by mutating theamino acid sequence at one or more furin cleavage sites such that themutation abolishes at least one furin cleavage site. Thus, in someembodiments, a furin-resistant GILT tag is a furin-resistant IGF-IImutein containing a mutation that abolishes at least one furin proteasecleavage site or changes a sequence adjacent to the furin proteasecleavage site such that the furin cleavage is prevented, inhibited,reduced or slowed down as compared to a wild-type IGF-II peptide (e.g.,wild-type human mature IGF-II). Typically, a suitable mutation does notimpact the ability of the furin-resistant GILT tag to bind to the humancation-independent mannose-6-phosphate receptor. In particular, afurin-resistant IGF-II mutein suitable for the invention binds to thehuman cation-independent mannose-6-phosphate receptor in amannose-6-phosphate-independent manner with a dissociation constant of10⁻⁷M or less (e.g., 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, or less) at pH 7.4. Insome embodiments, a furin-resistant IGF-II mutein contains a mutationwithin a region corresponding to amino acids 30-40 (e.g., 30-40, 31-40,32-40, 33-40, 34-40, 30-39, 31-39, 32-39, 34-37, 33-39, 34-39, 35-39,36-39, 37-40) of mature human IGF-II. In some embodiments, a suitablemutation abolishes at least one furin protease cleavage site. A mutationcan be amino acid substitutions, deletions, insertions. For example, anyone amino acid within the region corresponding to residues 30-40 (e.g.,30-40, 31-40, 32-40, 33-40, 34-40, 30-39, 31-39, 32-39, 34-37, 33-39,34-39, 35-39, 36-39, 37-40) of SEQ ID NO:5 can be substituted with anyother amino acid or deleted. For example, substitutions at position 34may affect furin recognition of the first cleavage site. Insertion ofone or more additional amino acids within each recognition site mayabolish one or both furin cleavage sites. Deletion of one or more of theresidues in the degenerate positions may also abolish both furincleavage sites.

In various embodiments, a furin-resistant IGF-II mutein contains aminoacid substitutions at positions corresponding to Arg37 or Arg40 ofmature human IGF-II. In some embodiments, a furin-resistant IGF-IImutein contains a Lys or Ala substitution at positions Arg37 or Arg40.Other substitutions are possible, including combinations of Lys and/orAla mutations at both positions 37 and 40, or substitutions of aminoacids other than Lys or Ala.

In various embodiments, an IGF-II mutein suitable for use herein maycontain additional mutations. For example, up to 30% or more of theresidues of SEQ ID NO:1 may be changed (e.g., up to 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% or more residues may bechanged). Thus, an IGF-II mutein suitable for use herein may have anamino acid sequence at least 70%, including at least 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to mature human IGF-II.

In various embodiments, an IGF-II mutein suitable for use herein istargeted specifically to the CI-MPR. Particularly useful are mutationsin the IGF-II polypeptide that result in a protein that binds the CI-MPRwith high affinity (e.g., with a dissociation constant of 10⁻⁷M or lessat pH 7.4) while binding other receptors known to be bound by IGF-IIwith reduced affinity relative to native IGF-II. For example, afurin-resistant IGF-II mutein suitable for the invention can be modifiedto have diminished binding affinity for the IGF-I receptor relative tothe affinity of naturally-occurring human IGF-II for the IGF-I receptor.For example, substitution of IGF-II residues Tyr 27 with Leu, Leu 43with Val or Ser 26 with Phe diminishes the affinity of IGF-II for theIGF-I receptor by 94-, 56-, and 4-fold respectively (Tones et al. (1995)J. Mol. Biol. 248(2):385-401). Deletion of residues 1-7 of human IGF-IIresulted in a 30-fold decrease in affinity for the human IGF-I receptorand a concomitant 12 fold increase in affinity for the rat IGF-IIreceptor (Hashimoto et al. (1995) J. Biol. Chem. 270(30):18013-8). TheNMR structure of IGF-II shows that Thr-7 is located near residues Phe-48Phe and Ser-50 as well as near the Cys-9-Cys-47 disulfide bridge. It isthought that interaction of Thr-7 with these residues can stabilize theflexible N-terminal hexapeptide required for IGF-I receptor binding(Terasawa et al. (1994) EMBO J. 13(23)5590-7). At the same time thisinteraction can modulate binding to the IGF-II receptor. Truncation ofthe C-terminus of IGF-II (residues 62-67) also appear to lower theaffinity of IGF-II for the IGF-I receptor by 5 fold (Roth et al. (1991)Biochem. Biophys. Res. Commun. 181(2):907-14).

The binding surfaces for the IGF-I and cation-independent M6P receptorsare on separate faces of IGF-II. Based on structural and mutationaldata, functional cation-independent M6P binding domains can beconstructed that are substantially smaller than human IGF-II. Forexample, the amino terminal amino acids (e.g., 1-7 or 2-7) and/or thecarboxy terminal residues 62-67 can be deleted or replaced.Additionally, amino acids 29-40 can likely be eliminated or replacedwithout altering the folding of the remainder of the polypeptide orbinding to the cation-independent M6P receptor. Thus, a targeting moietyincluding amino acids 8-28 and 41-61 can be constructed. These stretchesof amino acids could perhaps be joined directly or separated by alinker. Alternatively, amino acids 8-28 and 41-61 can be provided onseparate polypeptide chains. Comparable domains of insulin, which ishomologous to IGF-II and has a tertiary structure closely related to thestructure of IGF-II, have sufficient structural information to permitproper refolding into the appropriate tertiary structure, even whenpresent in separate polypeptide chains (Wang et al. (1991) TrendsBiochem. Sci. 279-281). Thus, for example, amino acids 8-28, or aconservative substitution variant thereof, could be fused to a lysosomalenzyme; the resulting fusion protein could be admixed with amino acids41-61, or a conservative substitution variant thereof, and administeredto a patient.

IGF-II can also be modified to minimize binding to serum IGF-bindingproteins (Baxter (2000) Am. J. Physiol Endocrinol Metab. 278(6):967-76)to avoid sequestration of IGF-II/GILT constructs. A number of studieshave localized residues in IGF-II necessary for binding to IGF-bindingproteins. Constructs with mutations at these residues can be screenedfor retention of high affinity binding to the M6P/IGF-II receptor andfor reduced affinity for IGF-binding proteins. For example, replacingPhe-26 of IGF-II with Ser is reported to reduce affinity of IGF-II forIGFBP-1 and -6 with no effect on binding to the M6P/IGF-II receptor(Bach et al. (1993) J. Biol. Chem. 268(13):9246-54). Othersubstitutions, such as Lys for Glu-9, can also be advantageous. Theanalogous mutations, separately or in combination, in a region of IGF-Ithat is highly conserved with IGF-II result in large decreases in IGF-BPbinding (Magee et al. (1999) Biochemistry 38(48):15863-70).

An alternate approach is to identify minimal regions of IGF-II that canbind with high affinity to the M6P/IGF-II receptor. The residues thathave been implicated in IGF-II binding to the M6P/IGF-II receptor mostlycluster on one face of IGF-II (Terasawa et al. (1994) EMBO J.13(23):5590-7). Although IGF-II tertiary structure is normallymaintained by three intramolecular disulfide bonds, a peptideincorporating the amino acid sequence on the M6P/IGF-II receptor bindingsurface of IGF-II can be designed to fold properly and have bindingactivity. Such a minimal binding peptide is a highly preferred lysosomaltargeting domain. For example, a preferred lysosomal targeting domain isamino acids 8-67 of human IGF-II. Designed peptides, based on the regionaround amino acids 48-55, which bind to the M6P/IGF-II receptor, arealso desirable lysosomal targeting domains. Alternatively, a randomlibrary of peptides can be screened for the ability to bind theM6P/IGF-II receptor either via a yeast two hybrid assay, or via a phagedisplay type assay.

Binding Affinity for the Insulin Receptor

Many IGF-II muteins, including furin-resistant IGF-II muteins, describedherein have reduced or diminished binding affinity for the insulinreceptor. Thus, in some embodiments, a peptide tag suitable for theinvention has reduced or diminished binding affinity for the insulinreceptor relative to the affinity of naturally-occurring human IGF-IIfor the insulin receptor. In some embodiments, peptide tags with reducedor diminished binding affinity for the insulin receptor suitable for theinvention include peptide tags having a binding affinity for the insulinreceptor that is more than 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold,18-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold,90-fold, or 100-fold less than that of the wild-type mature humanIGF-II. The binding affinity for the insulin receptor can be measuredusing various in vitro and in vivo assays known in the art. Exemplarybinding assays are described in the Examples section.

Mutagenesis

IGF-II muteins can be prepared by introducing appropriate nucleotidechanges into the IGF-II DNA, or by synthesis of the desired IGF-IIpolypeptide. Variations in the IGF-II sequence can be made, for example,using any of the techniques and guidelines for conservative andnon-conservative mutations set forth, for instance, in U.S. Pat. No.5,364,934. Variations may be a substitution, deletion or insertion ofone or more codons encoding IGF-II that results in a change in the aminoacid sequence of IGF-II as compared with a naturally-occurring sequenceof mature human IGF-II. Amino acid substitutions can be the result ofreplacing one amino acid with another amino acid having similarstructural and/or chemical properties, such as the replacement of aleucine with a serine, i.e., conservative amino acid replacements. Aminoacid substitutions can also be the result of replacing one amino acidwith another amino acid having dis-similar structural and/or chemicalproperties, i.e., non-conservative amino acid replacements. Insertionsor deletions may optionally be in the range of 1 to 5 amino acids. Thevariation allowed may be determined by systematically making insertions,deletions or substitutions of amino acids in the sequence and testingthe resulting variants for activity in the in vivo or in vitro assaysknown in the art (such as binding assays to the CI-MPR or furin cleavageassays).

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant. Alanine is alsotypically preferred because it is the most common amino acid. Further,it is frequently found in both buried and exposed positions [Creighton,The Proteins, (W. H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1(1976)]. If alanine substitution does not yield adequate amounts ofvariant, an isoteric amino acid can be used.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce IGF-II muteins.

Spacer

A GILT tag can be fused to the N-terminus or C-terminus of a lysosomalenzyme. The GILT tag can be fused directly to the lysosomal enzyme orcan be separated from the lysosomal enzyme by a linker or a spacer. Anamino acid linker or spacer is generally designed to be rigid, flexibleor to interpose a structure, such as an alpha-helix, between the twoprotein moieties. A linker or spacer can be relatively short, such as,for example, the sequence Gly-Ala-Pro (GAP) (SEQ ID NO: 9),Gly-Gly-Gly-Gly-Ala (GGGGA) (SEQ ID NO: 60) or Gly-Gly-Gly-Gly-Ser(GGGGS) (SEQ ID NO: 12), or can be longer, such as, for example, 10-25amino acids in length, 25-50 amino acids in length or 35-55 amino acidsin length. The site of a fusion junction should be selected with care topromote proper folding and activity of both fusion partners and toprevent premature separation of a peptide tag from the lysosomal enzyme,e.g., alpha-N-acetylglucosaminidase.

In various embodiments, the spacer peptide comprises one or more GGGPS(SEQ ID NO: 14) or GGGSP (SEQ ID NO: 15) amino acid sequences, andoptionally further comprises one or more of (i) GAP (SEQ ID NO: 9), (ii)GGGGS (SEQ ID NO: 12), (iii) GGGS (SEQ ID NO: 16), (iv) AAAAS (SEQ IDNO: 17), (v) AAAS (SEQ ID NO: 18), (vi) PAPA (SEQ ID NO: 19), (vii)TPAPA (SEQ ID NO: 20), (viii) AAAKE (SEQ ID NO: 21) or (ix) GGGGA (SEQID NO: 60). In various embodiments, the spacer comprises the amino acidsequence GAP (SEQ ID NO: 9), GPS (SEQ ID NO: 10), or GGS (SEQ ID NO:11).

In various embodiments, the spacer peptide comprises one or more GGGGS(SEQ ID NO: 12) or GGGS (SEQ ID NO: 16) amino acid sequences. In variousembodiments, the spacer peptide comprises one or more GGGPS (SEQ ID NO:14) or GGGSP (SEQ ID NO: 15) amino acid sequences. In variousembodiments, the spacer peptide comprises one or more AAAAS (SEQ ID NO:17) or AAAS (SEQ ID NO: 18) amino acid sequences. In variousembodiments, the spacer peptide comprises one or more PAPA (SEQ ID NO:19) or TPAPA (SEQ ID NO: 20) amino acid sequences. In variousembodiments, the spacer peptide comprises one or more AAAKE (SEQ ID NO:21) amino acid sequences. In various embodiments, the spacer peptidecomprises one or more GGGGA (SEQ ID NO: 60) amino acid sequences.

In various embodiments, the spacer peptide comprises an amino acidsequence selected from the group consisting of:

(SEQ ID NOs: 12, 56, 58, 91-94) (GGGGS)_(n), (SEQ ID NOs: 36, 95-100)(GGGGS)_(n)-GGGPS, (SEQ ID NOs: 101-107) GAP-(GGGGS)_(n)-GGGPS,(SEQ ID NOs: 37, 108-113) GAP-(GGGGS)_(n)-GGGPS-GAP,(SEQ ID NOs: 114-162) GAP-(GGGGS)_(n)-GGGPS-(GGGGS)_(n)-GAP,(SEQ ID NOs: 163-169) GAP-GGGPS-(GGGGS)_(n)-GAP, (SEQ ID NOs: 170-218)GAP-(GGGGS)_(n)-AAAAS-GGGPS-(GGGGS)_(n)-AAAA-GAP, (SEQ ID NOs: 219-267)GAP-(GGGGS)_(n)-PAPAP-(Xaa)_(n)-GAP, (SEQ ID NOs: 268-316)GAP-(GGGGS)_(n)-PAPAPT-(Xaa)_(n)-GAP, (SEQ ID NOs: 544-551)GAP-(GGGGS)_(n)-(Xaa)n-PAPAP-(Xaa)n-(AAAKE)n-(Xaa)n- (GGGGS)_(n)-GAP,(SEQ ID NOs: 60, 79, 81, 317-320) (GGGGA)_(n), (SEQ ID NOs: 321-326)(GGGGA)_(n)-GGGPS, (SEQ ID NOs: 327-333) GAP-(GGGGA)_(n)-GGGPS,(SEQ ID NOs: 334-340) GAP-(GGGGA)_(n)-GGGPS-GAP, (SEQ ID NOs: 341-389)GAP-(GGGGA)_(n)-GGGPS-(GGGGA)_(n)-GAP, (SEQ ID NOs: 390-396)GAP-GGGPS-(GGGGA)_(n)-GAP, (SEQ ID NOs: 397-445)GAP-(GGGGA)_(n)-AAAAS-GGGPS-(GGGGA)_(n)-AAAA-GAP, (SEQ ID NOs: 446-494)GAP-(GGGGA)_(n)-PAPAP-(Xaa)_(n)-GAP, (SEQ ID NOs: 495-543)GAP-(GGGGA)_(n)-PAPAPT-(Xaa)_(n)-GAP, (SEQ ID NOs: 552-559)GAP-(GGGGA)_(n)-(Xaa)_(n)-PAPAP-(Xaa)_(n)-(AAAKE)n-(Xaa)_(n)-(GGGGA)_(n)-GAP; wherein n is 1 to 7. In various embodiments, n is1 to 4.

In various embodiments, the spacer is selected from the group consistingof

(SEQ ID NO: 22) EFGGGGSTR, (SEQ ID NO: 9) GAP, (SEQ ID NO: 12) GGGGS,(SEQ ID NO: 23) GPSGSPG, (SEQ ID NO: 24) GPSGSPGT, (SEQ ID NO: 25)GPSGSPGH, (SEQ ID NO: 26) GGGGSGGGGSGGGGSGGGGSGGGPST, (SEQ ID NO: 27)GGGGSGGGGSGGGGSGGGGSGGGPSH, (SEQ ID NO: 28)GGGGSGGGGSGGGGSGGGGSGGGPSGGGGSGGGPS, (SEQ ID NO: 29)GAPGGGGSGGGGSGGGGSGGGGSGGGPSGGGGSGGGPSGAP, (SEQ ID NO: 30)GGGGSGGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGGSGGGGSGGGP S, (SEQ ID NO: 31)GAPGGGGSGGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGGSGGGGSGG GPSGAP,(SEQ ID NO: 32) GGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGPS, (SEQ ID NO: 33)GAPGGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGPSGAP, (SEQ ID NO: 34)GGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGPSGGGGSGGGGSGGGP S, (SEQ ID NO: 35)GAPGGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGPSGGGGSGGGGSGG GPSGAP,(SEQ ID NO: 36) GGGGSGGGGSGGGGSGGGGSGGGPS, (SEQ ID NO: 37)GAPGGGGSGGGGSGGGGSGGGGSGGGPSGAP, (SEQ ID NO: 38)GGGGSGGGGSAAAASGGGGSGGGPS, (SEQ ID NO: 39)GAPGGGGSGGGGSAAAASGGGGSGGGPSGAP, (SEQ ID NO: 40)GGGGSGGGGSAAAASGGGGSGGGGSAAAASGGGGSGGGGSAAAASGGGP S, (SEQ ID NO: 41)GAPGGGGSGGGGSAAAASGGGGSGGGGSAAAASGGGGSGGGGSAAAASGG GPSGAP,(SEQ ID NO: 42) GGGGSGGGGSAAAASGGGPSGGGGSAAAASGGGPSGGGGSAAAASGGGP S,(SEQ ID NO: 43) GAPGGGGSGGGGSAAAASGGGPSGGGGSAAAASGGGPSGGGGSAAAASGGGPSGAP, (SEQ ID NO: 44) GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPGPS,(SEQ ID NO: 45) GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPGPS GAP,(SEQ ID NO: 46) GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTGP S,(SEQ ID NO: 47) GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTGPSGAP, (SEQ ID NO: 48) GGGSPAPTPTPAPTPAPTPAGGGPS, (SEQ ID NO: 49)GAPGGGSPAPTPTPAPTPAPTPAGGGPSGAP, (SEQ ID NO: 50)GGGSPAPAPTPAPAPTPAPAGGGPS, (SEQ ID NO: 51)GAPGGGSPAPAPTPAPAPTPAPAGGGPSGAP, (SEQ ID NO: 52)GGGSAEAAAKEAAAKEAAAKAGGPS, (SEQ ID NO: 53)GAPGGGSAEAAAKEAAAKEAAAKAGGPSGAP, (SEQ ID NO: 54)GGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGG, (SEQ ID NO: 55)GAPGGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGGGAP, (SEQ ID NO: 56)GGGGSGGGGSGGGGS, (SEQ ID NO: 57) GAPGGGGSGGGGSGGGGSGAP, (SEQ ID NO: 58)GGGGSGGGGSGGGGSGGGGS, (SEQ ID NO: 59) GAPGGGGSGGGGSGGGGSGGGGSGAP,(SEQ ID NO: 60) GGGGA, (SEQ ID NO: 61) GGGGAGGGGAGGGGAGGGGAGGGPST,(SEQ ID NO: 62) GGGGAGGGGAGGGGAGGGGAGGGPSH, (SEQ ID NO: 63)GGGGAGGGGAGGGGAGGGGAGGGPSGGGGAGGGPS, (SEQ ID NO: 64)GAPGGGGAGGGGAGGGGAGGGGAGGGPSGGGGAGGGPSGAP, (SEQ ID NO: 65)GGGGAGGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGGAGGGGAGGGP S, (SEQ ID NO: 66)GAPGGGGAGGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGGAGGGGAGG GPSGAP,(SEQ ID NO: 67) GGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGPS, (SEQ ID NO: 68)GAPGGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGPSGAP, (SEQ ID NO: 69)GGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGPSGGGGAGGGGAGGGP S, (SEQ ID NO: 70)GAPGGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGPSGGGGAGGGGAGG GPSGAP,(SEQ ID NO: 71) GGGGAGGGGAGGGGAGGGGAGGGPS, (SEQ ID NO: 72)GAPGGGGAGGGGAGGGGAGGGGAGGGPSGAP, (SEQ ID NO: 73)GGGGAGGGGAAAAASGGGGAGGGPS, (SEQ ID NO: 74)GAPGGGGAGGGGAAAAASGGGGAGGGPSGAP, (SEQ ID NO: 75)GGGGAGGGGAAAAASGGGGAGGGGAAAAASGGGGAGGGGAAAAASGGGP S, (SEQ ID NO: 76)GAPGGGGAGGGGAAAAASGGGGAGGGGAAAAASGGGGAGGGGAAAAASGG GPSGAP,(SEQ ID NO: 77) GGGGAGGGGAAAAASGGGPSGGGGAAAAASGGGPSGGGGAAAAASGGGP S,(SEQ ID NO: 78) GAPGGGGAGGGGAAAAASGGGPSGGGGAAAAASGGGPSGGGGAAAAASGGGPSGAP, (SEQ ID NO: 79) GGGGAGGGGAGGGGA, (SEQ ID NO: 80)GAPGGGGAGGGGAGGGGAGAP, (SEQ ID NO: 81) GGGGAGGGGAGGGGAGGGGA,(SEQ ID NO: 82) GAPGGGGAGGGGAGGGGAGGGGAGAP, (SEQ ID NO: 83)GGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGPS [or (GGGGA)8GGGPS],(SEQ ID NO: 84) GGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGPSH[or (GGGGA)8GGGPSH], (SEQ ID NO: 85)GGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGPS [or (GGGGA)9GGGPS],(SEQ ID NO: 86) GGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGPSH [or (GGGGA)9GGGPSH], (SEQ ID NO: 87) GGGGPAPGPGPAPGPAPGPAGGGPS,(SEQ ID NO: 88) GAPGGGGPAPGPGPAPGPAPGPAGGGPGGAP, (SEQ ID NO: 89)GGGGPAPAPGPAPAPGPAPAGGGPS, and (SEQ ID NO: 90)GAPGGGGPAPAPGPAPAPGPAPAGGGPGGAP.

In various embodiments, the spacer is selected from the group consistingof

(SEQ ID NO: 36) GGGGSGGGGSGGGGSGGGGSGGGPS, (SEQ ID NO: 44)GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPGPS, (SEQ ID NO: 45)GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPGPS GAP, (SEQ ID NO: 46)GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTGP S, (SEQ ID NO: 47)GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST GPSGAP,(SEQ ID NO: 48) GGGSPAPTPTPAPTPAPTPAGGGPS, (SEQ ID NO: 49)GAPGGGSPAPTPTPAPTPAPTPAGGGPSGAP, (SEQ ID NO: 50)GGGSPAPAPTPAPAPTPAPAGGGPS, (SEQ ID NO: 51)GAPGGGSPAPAPTPAPAPTPAPAGGGPSGAP, (SEQ ID NO: 52)GGGSAEAAAKEAAAKEAAAKAGGPS, (SEQ ID NO: 53)GAPGGGSAEAAAKEAAAKEAAAKAGGPSGAP, (SEQ ID NO: 54)GGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGG, (SEQ ID NO: 55)GAPGGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGGGAP, and (SEQ ID NO: 71)GGGGAGGGGAGGGGAGGGGAGGGPS,

In various embodiments, the spacer is selected from the group consistingof

(SEQ ID NO: 36) GGGGSGGGGSGGGGSGGGGSGGGPS, (SEQ ID NO: 47)GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST GPSGAP,(SEQ ID NO: 51) GAPGGGSPAPAPTPAPAPTPAPAGGGPSGAP, (SEQ ID NO: 55)GAPGGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGGGAP, and (SEQ ID NO: 71)GGGGAGGGGAGGGGAGGGGAGGGPS.

Additional constructs of GILT-tagged alpha-N-acetylglucosaminidaseproteins that can be used in the methods and compositions of the presentinvention were described in detail in U.S. Publication Nos. 20050244400and 20050281805, the entire disclosures of which is incorporated hereinby reference.

Cells

Any mammalian cell or cell type susceptible to cell culture, and toexpression of polypeptides, may be utilized in accordance with thepresent invention, such as, for example, human embryonic kidney (HEK)293, Chinese hamster ovary (CHO), monkey kidney (COS), HT1080, C10,HeLa, baby hamster kidney (BHK), 3T3, C127, CV-1, HaK, NS/0, and L-929cells. Non-limiting examples of mammalian cells that may be used inaccordance with the present invention include, but are not limited to,BALB/c mouse myeloma line (NS0/1, ECACC No: 85110503); humanretinoblasts (PER.C6 (CruCell, Leiden, The Netherlands)); monkey kidneyCV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonickidney line (293 or 293 cells subcloned for growth in suspensionculture, Graham et al., J. Gen Virol., 36:59 (1977)); baby hamsterkidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells +/−DHFR(CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980));mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980));monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells(VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCCCCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells(BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); humanliver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCCCCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68(1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). Insome embodiments, the fusion protein of the present invention isproduced from CHO cell lines.

The fusion protein of the invention can also be expressed in a varietyof non-mammalian host cells such as, for example, insect (e.g., Sf-9,Sf-21, Hi5), plant (e.g., Leguminosa, cereal, or tobacco), yeast (e.g.,S. cerivisae, P. pastoris), prokaryote (e.g., E. Coli, B. subtilis andother Bacillus spp., Pseudomonas spp., Streptomyces spp), or fungus.

In some embodiments, a fusion protein with or without a furin-resistantGILT tag can be produced in furin-deficient cells. As used herein, theterm “furin-deficient cells” refers to any cells whose furin proteaseactivity is inhibited, reduced or eliminated. Furin-deficient cellsinclude both mammalian and non-mammalian cells that do not produce furinor produce reduced amount or defective furin protease. Exemplary furindeficient cells that are known and available to the skilled artisan,including but not limited to FD11 cells (Gordon et al (1997) Infectionand Immunity 65(8):3370 3375), and those mutant cells described inMoebring and Moehring (1983) Infection and Immunity 41(3):998 1009.Alternatively, a furin deficient cell may be obtained by exposing theabove-described mammalian and non-mammalian cells to mutagenesistreatment, e.g., irradiation, ethidium bromide, bromidated uridine(BrdU) and others, preferably chemical mutagenesis, and more preferredethyl methane sulfonate mutagenesis, recovering the cells which survivethe treatment and selecting for those cells which are found to beresistant to the toxicity of Pseudomonas exotoxin A (see Moehring andMoehrin (1983) Infection and Immunity 41(3):998 1009).

In various embodiments, it is contemplated that certain of the targetedtherapeutic proteins comprising a spacer as described herein may exhibitincreased expression of active protein when expressed recombinantlycompared to targeted therapeutic proteins comprising a different spacerpeptide. In various embodiments, it is also contemplated that targetedtherapeutic proteins described herein may have increased activitycompared to other targeted therapeutic proteins herein. It iscontemplated that those targeted therapeutic proteins exhibitingincreased expression of active protein and/or having increased activitycompared to other targeted therapeutic proteins comprising a differentspacer peptide are used for further experimentation.

Administration of Therapeutic Proteins

In accordance of the invention, a therapeutic protein of the inventionis typically administered to the individual alone, or in compositions ormedicaments comprising the therapeutic protein (e.g., in the manufactureof a medicament for the treatment of the disease), as described herein.The compositions can be formulated with a physiologically acceptablecarrier or excipient to prepare a pharmaceutical composition. Thecarrier and composition can be sterile. The formulation should suit themode of administration.

Suitable pharmaceutically acceptable carriers include but are notlimited to water, salt solutions (e.g., NaCl), saline, buffered saline,alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzylalcohols, polyethylene glycols, gelatin, carbohydrates such as lactose,amylose or starch, sugars such as mannitol, sucrose, or others,dextrose, magnesium stearate, talc, silicic acid, viscous paraffin,perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinylpyrolidone, etc., as well as combinations thereof. The pharmaceuticalpreparations can, if desired, be mixed with auxiliary agents (e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, coloring, flavoringand/or aromatic substances and the like) which do not deleteriouslyreact with the active compounds or interference with their activity. Ina preferred embodiment, a water-soluble carrier suitable for intravenousadministration is used.

The composition or medicament, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thecomposition can be a liquid solution, suspension, emulsion, tablet,pill, capsule, sustained release formulation, or powder. The compositioncan also be formulated as a suppository, with traditional binders andcarriers such as triglycerides. Oral formulation can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose,magnesium carbonate, etc.

The composition or medicament can be formulated in accordance with theroutine procedures as a pharmaceutical composition adapted foradministration to human beings. For example, in a preferred embodiment,a composition for intravenous administration typically is a solution insterile isotonic aqueous buffer. Where necessary, the composition mayalso include a solubilizing agent and a local anesthetic to ease pain atthe site of the injection. Generally, the ingredients are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampule or sachette indicating the quantityof active agent. Where the composition is to be administered byinfusion, it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water, saline or dextrose/water. Where thecomposition is administered by injection, an ampule of sterile water forinjection or saline can be provided so that the ingredients may be mixedprior to administration.

The therapeutic protein can be formulated as neutral or salt forms.Pharmaceutically acceptable salts include those formed with free aminogroups such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with free carboxyl groupssuch as those derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

A therapeutic protein (or a composition or medicament containing atherapeutic protein) is administered by any appropriate route. Invarious embodiments, a therapeutic protein is administeredintravenously. In other embodiments, a therapeutic protein isadministered by direct administration to a target tissue, such as heartor muscle (e.g., intramuscular), or nervous system (e.g., directinjection into the brain; intraventricularly; intrathecally). In variousembodiments, a therapeutic protein is administered intrathecally.Alternatively, a therapeutic protein (or a composition or medicamentcontaining a therapeutic protein) can be administered parenterally,transdermally, or transmucosally (e.g., orally or nasally). More thanone route can be used concurrently, if desired, e.g., a therapeuticprotein is administered intravenously and intrathecally. Concurrentintravenous and intrathecal administration need not be simultaneous, butcan be sequential.

A therapeutic protein (or a composition or medicament containing atherapeutic protein) can be administered alone, or in conjunction withother agents, such as antihistamines (e.g., diphenhydramine) orimmunosuppressants or other immunotherapeutic agents which counteractanti-GILT-tagged lysosomal enzyme antibodies. The term, “in conjunctionwith, “indicates that the agent is administered prior to, at about thesame time as, or following the therapeutic protein (or a composition ormedicament containing the therapeutic protein). For example, the agentcan be mixed into a composition containing the therapeutic protein, andthereby administered contemporaneously with the therapeutic protein;alternatively, the agent can be administered contemporaneously, withoutmixing (e.g., by “piggybacking” delivery of the agent on the intravenousline by which the therapeutic protein is also administered, or viceversa). In another example, the agent can be administered separately(e.g., not admixed), but within a short time frame (e.g., within 24hours) of administration of the therapeutic protein.

The therapeutic protein (or composition or medicament containing thetherapeutic protein) is administered in a therapeutically effectiveamount (i.e., a dosage amount that, when administered at regularintervals, is sufficient to treat the disease, such as by amelioratingsymptoms associated with the disease, preventing or delaying the onsetof the disease, and/or also lessening the severity or frequency ofsymptoms of the disease, as described above). The dose which will betherapeutically effective for the treatment of the disease will dependon the nature and extent of the disease's effects, and can be determinedby standard clinical techniques. In addition, in vitro or in vivo assaysmay optionally be employed to help identify optimal dosage ranges usingmethods known in the art. The precise dose to be employed will alsodepend on the route of administration, and the seriousness of thedisease, and should be decided according to the judgment of apractitioner and each patient's circumstances. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems. The therapeutically effective dosage amount can be,for example, about 0.1-1 mg/kg, about 1-5 mg/kg, about 2.5-20 mg/kg,about 5-20 mg/kg, about 20-50 mg/kg, or about 20-100 mg/kg or about50-200 mg/kg, or about 2.5 to 20 mg/kg of body weight. The effectivedose for a particular individual can be varied (e.g., increased ordecreased) over time, depending on the needs of the individual. Forexample, in times of physical illness or stress, or if disease symptomsworsen, the dosage amount can be increased.

The therapeutically effective amount of the therapeutic protein (orcomposition or medicament containing the therapeutic protein) isadministered at regular intervals, depending on the nature and extent ofthe disease's effects, and on an ongoing basis. Administration at an“interval,” as used herein, indicates that the therapeutically effectiveamount is administered periodically (as distinguished from a one-timedose). The interval can be determined by standard clinical techniques.In some embodiments, the therapeutic protein is administered bimonthly,monthly, twice monthly, triweekly, biweekly, weekly, twice weekly,thrice weekly, or daily. The administration interval for a singleindividual need not be a fixed interval, but can be varied over time,depending on the needs of the individual. For example, in times ofphysical illness or stress, or if disease symptoms worsen, the intervalbetween doses can be decreased.

As used herein, the term “bimonthly” means administration once per twomonths (i.e., once every two months); the term “monthly” meansadministration once per month; the term “triweekly” means administrationonce per three weeks (i.e., once every three weeks); the term “biweekly”means administration once per two weeks (i.e., once every two weeks);the term “weekly” means administration once per week; and the term“daily” means administration once per day.

The disclosure additionally pertains to a pharmaceutical compositioncomprising a therapeutic protein, as described herein, in a container(e.g., a vial, bottle, bag for intravenous administration, syringe,etc.) with a label containing instructions for administration of thecomposition for treatment of Mucopolysaccharidosis Type IIIB (SanfilippoB Syndrome), such as by the methods described herein.

Intrathecal Administration of the Pharmaceutically AcceptableFormulations

In various embodiments, the enzyme fusion protein is administered byintroduction into the central nervous system of the subject, e.g., intothe cerebrospinal fluid of the subject. In certain aspects of theinvention, the enzyme is introduced intrathecally, e.g., into the lumbararea, or the cistema magna or intraventricularly (orintracerebroventricularly) into a cerebral ventricle space. Methods ofadministering a lysosomal enzyme intrathecally are described in U.S.Pat. No. 7,442,372, incorporated herein by reference in its entirety.

Those of skill in the art are aware of devices that may be used toeffect intrathecal administration of a therapeutic composition. Forexample, the therapy may be given using an Ommaya reservoir which is incommon use for intrathecally administering drugs for meningealcarcinomatosis (Ommaya A K, Lancet 2: 983-84, 1963). More specifically,in this method, a ventricular tube is inserted through a hole formed inthe anterior horn and is connected to an Ommaya reservoir installedunder the scalp, and the reservoir is subcutaneously punctured tointrathecally deliver the particular enzyme being replaced, which isinjected into the reservoir. Other devices for intrathecaladministration of therapeutic compositions to an individual aredescribed in U.S. Pat. No. 6,217,552, incorporated herein by reference.Alternatively, the composition may be intrathecally given, for example,by a single injection, or continuous infusion. It should be understoodthat the dosage treatment may be in the form of a single doseadministration or multiple doses.

As used herein, the term “intrathecal administration” is intended toinclude delivering a pharmaceutical composition directly into thecerebrospinal fluid of a subject, by techniques including lateralcerebroventricular injection (i.e., intracerebroventricularly) through aburrhole or cistemal or lumbar puncture or the like (described inLazorthes et al. Advances in Drug Delivery Systems and Applications inNeurosurgery, 143-192 and Omaya et al., Cancer Drug Delivery, 1:169-179, the contents of which are incorporated herein by reference).The term “lumbar region” is intended to include the area between thethird and fourth lumbar (lower back) vertebrae and, more inclusively,the L2-S1 region of the spine. The term “cisterna magna” is intended toinclude access to the space around and below the cerebellum via theopening between the skull and the top of the spine. The term “cerebralventricle” is intended to include the cavities in the brain that arecontinuous with the central canal of the spinal cord. Administration ofa pharmaceutical composition in accordance with the present invention toany of the above mentioned sites can be achieved by direct injection ofthe composition or by the use of infusion pumps. For injection, thecomposition of the invention can be formulated in liquid solutions,preferably in physiologically compatible buffers such as Hank'ssolution, Ringer's solution or phosphate buffer. In addition, the enzymemay be formulated in solid form and re-dissolved or suspendedimmediately prior to use. Lyophilized forms are also included. Theinjection can be, for example, in the form of a bolus injection orcontinuous infusion (e.g., using infusion pumps) of the enzyme.

In various embodiments of the invention, the enzyme is administered bylateral cerebro ventricular injection into the brain of a subject. Theinjection can be made, for example, through a burr hole made in thesubject's skull. In another embodiment, the enzyme and/or otherpharmaceutical formulation is administered through a surgically insertedshunt into the cerebral ventricle of a subject. For example, theinjection can be made into the lateral ventricles, which are larger,even though injection into the third and fourth smaller ventricles canalso be made.

In various embodiments, the pharmaceutical compositions used in thepresent invention are administered by injection into the cisterna magna,or lumbar area of a subject. In another embodiment of the method of theinvention, the pharmaceutically acceptable formulation providessustained delivery, e.g., “slow release” of the enzyme or otherpharmaceutical composition used in the present invention, to a subjectfor at least one, two, three, four weeks or longer periods of time afterthe pharmaceutically acceptable formulation is administered to thesubject.

In various embodiments, a therapeutic fusion protein is delivered to oneor more surface or shallow tissues of the brain or spinal cord. Forexample, in various embodiments, a therapeutic fusion protein isdelivered to one or more surface or shallow tissues of the cerebrum orspinal cord. In some embodiments, the targeted surface or shallowtissues of the cerebrum or spinal cord are located within 4 mm from thesurface of the cerebrum. In some embodiments, the targeted surface orshallow tissues of the cerebrum are selected from pia mater tissues,cerebral cortical ribbon tissues, hippocampus, Virchow Robin space,blood vessels within the VR space, the hippocampus, portions of thehypothalamus on the inferior surface of the brain, the optic nerves andtracts, the olfactory bulb and projections, and combinations thereof.

In some embodiments, a therapeutic fusion protein is delivered to one ormore deep tissues of the cerebrum or spinal cord. In some embodiments,the targeted surface or shallow tissues of the cerebrum or spinal cordare located 4 mm (e.g., 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm) below(or internal to) the surface of the cerebrum. In some embodiments,targeted deep tissues of the cerebrum include the cerebral corticalribbon. In some embodiments, targeted deep tissues of the cerebruminclude one or more of the diencephalon (e.g., the hypothalamus,thalamus, prethalamus, subthalamus, etc.), metencephalon, lentiformnuclei, the basal ganglia, caudate, putamen, amygdala, globus pallidus,and combinations thereof.

In various embodiments, a targeted surface or shallow tissue of thespinal cord contains pia matter and/or the tracts of white matter. Invarious embodiments, a targeted deep tissue of the spinal cord containsspinal cord grey matter and/or ependymal cells. In some embodiments, atherapeutic fusion protein is delivered to neurons of the spinal cord.

In various embodiments, a therapeutic fusion protein is delivered to oneor more tissues of the cerebellum. In certain embodiments, the targetedone or more tissues of the cerebellum are selected from the groupconsisting of tissues of the molecular layer, tissues of the Purkinjecell layer, tissues of the Granular cell layer, cerebellar peduncles,and combination thereof. In some embodiments, therapeutic agents (e.g.,enzymes) are delivered to one or more deep tissues of the cerebellumincluding, but not limited to, tissues of the Purkinje cell layer,tissues of the Granular cell layer, deep cerebellar white matter tissue(e.g., deep relative to the Granular cell layer), and deep cerebellarnuclei tissue.

In various embodiments, a therapeutic fusion protein is delivered to oneor more tissues of the brainstem. In some embodiments, the targeted oneor more tissues of the brainstem include brain stem white matter tissueand/or brain stem nuclei tissue.

In various embodiments, a therapeutic fusion protein is delivered tovarious brain tissues including, but not limited to, gray matter, whitematter, periventricular areas, pia-arachnoid, meninges, neocortex,cerebellum, deep tissues in cerebral cortex, molecular layer,caudate/putamen region, midbrain, deep regions of the pons or medulla,and combinations thereof.

In various embodiments, a therapeutic fusion protein is delivered tovarious cells in the brain including, but not limited to, neurons, glialcells, perivascular cells and/or meningeal cells. In some embodiments, atherapeutic protein is delivered to oligodendrocytes of deep whitematter.

Kits for Use in the Methods of the Invention

The agents utilized in the methods of the invention may be provided in akit, which kit may further include instructions for use. Such a kit willcomprise a fusion protein as described herein comprising an enzyme foruse in the treatment of a lysosomal storage disease and a lysosomaltargeting moiety, usually in a dose and form suitable for administrationto the host. In various embodiments, the kit will usually comprise adevice for delivering the enzyme intrathecally.

A kit may also be provided for the conjugation of an antigen,particularly a polypeptide antigen, to a high uptake moiety, in order togenerate a therapeutic composition. For example, a moiety such as anIGF-II mutein, either conjugated to a linker suitable for linkingpolypeptides, as described above, may be provided. The high uptakemoiety may also be provided in an unconjugated form, in combination witha suitable linker, and instructions for use.

Another kit may comprise instructions for the intrathecal administrationof the therapeutic compositions of the present invention, in addition tothe therapeutic compositions. In certain embodiments, the kits of theinvention may comprise catheters or other devices for the intrathecaladministration of the enzyme replacement therapy that are preloaded withthe therapeutic compositions of the present invention. For example,catheters preloaded with 0.001-0.01 mg, 0.01-0.1 mg, 0.1-1.0 mg, 1.0-10mg, 10-100 mg, or more of a therapeutic fusion protein comprising alysosomal enzyme and lysosomal targeting moiety, such as Naglu andIGF-II mutein, in a pharmaceutically acceptable formulation arespecifically contemplated. Exemplary catheters may single use cathetersthat can be discarded after use. Alternatively, the preloaded cathetersmay be refillable and presented in kits that have appropriate amounts ofthe enzyme for refilling such catheters.

The invention will be further and more specifically described by thefollowing examples. Examples, however, are included for illustrationpurposes, not for limitation.

Example 1—Generation of Spacer Sequences

Lysosomal enzymes comprising GILT tags and spacers have been disclosedin US Patent Publication Nos. 20030082176, 20040006008, 20040005309, and20050281805. Alpha-N-acetylglucosaminidase (Naglu) fusion proteinscomprising spacer peptides are disclosed in US Patent Publication No.201120232021. Additional spacer peptides for use in targeted therapeuticfusion proteins comprising a lysosomal enzyme and a GILT tag weredeveloped as described below.

Spacers can be developed to link both IGF-II muteins and furin-resistantIGF-II muteins. Exemplary spacers include the following amino acidsequences:

(SEQ ID NO: 22) EFGGGGSTR (SEQ ID NO: 9) GAP, (SEQ ID NO: 12) GGGGS,(SEQ ID NO: 60) GGGGA, (SEQ ID NO: 23) GPSGSPG, (SEQ ID NO: 24)GPSGSPGT, (SEQ ID NO: 25) GPSGSPH, (SEQ ID NO: 36)GGGGSGGGGSGGGGSGGGGSGGGPS, (SEQ ID NO: 71) GGGGAGGGGAGGGGAGGGGAGGGPS,(SEQ ID NO: 26) GGGGSGGGGSGGGGSGGGGSGGGPST, and (SEQ ID NO: 27)GGGGSGGGGSGGGGSGGGGSGGGPSH, (SEQ ID NO: 62) GGGGAGGGGAGGGGAGGGGAGGGPSH.

Constructs comprising a spacer, full-length Naglu (including the signalsequence) and an IGF-II peptide were generated in which the spacersequence (EFGGGGSTR spacer (SEQ ID NO: 22), GAP spacer (SEQ ID NO: 9),GGGGS spacer (SEQ ID NO: 12), GPSGSPG spacer (SEQ ID NO: 23), orGGGGSGGGGSGGGGSGGGGSGGGPS spacer (SEQ ID NO: 36)) was inserted betweenfull-length Naglu and IGF2 8-67 R37A (SEQ ID NOs: 560-564).

Additional linkers were made based on the XTEN method as described inSchellenberger et al. (Nat Biotech 27:1186-1190, 2009). XTEN-likelinkers may provide a longer half-life for the generated fusion proteinas compared to other linkers. Exemplary spacers have the amino acidsequences GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPGPS (SEQ ID NO:44), GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPGPSGAP (SEQ ID NO:45), GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTGPS (SEQ ID NO: 46),and GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTGPSGAP (SEQ ID NO:47). The spacer can be inserted between Naglu and IGF-2 mutein,optionally via the AscI sites on the constructs.

Protein expression has been associated with the DNA codon used to encodea particular amino acid, e.g., changing the codon for an amino acid canincrease expression of the protein without changing the amino acidsequence of the protein (Trinh et al, Mol. Immunol 40:717-722, 2004).Altering the codon encoding the peptide resulted in increased levels ofrecombinant fusion protein production. Using this technique additionalspacer sequences were developed for use in the therapeutic fusionprotein with lysosomal enzyme, such as GGGGSGGGGSGGGGS (SEQ ID NO: 56),GAPGGGGSGGGGSGGGGSGAP (SEQ ID NO: 57), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:58) and GAPGGGGSGGGGSGGGGSGGGGSGAP (SEQ ID NO: 59). Additional spacersequences are GGGGAGGGGAGGGGA (SEQ ID NO: 79), GAPGGGGAGGGGAGGGGAGAP(SEQ ID NO: 80), GGGGAGGGGAGGGGAGGGGA (SEQ ID NO: 81) andGAPGGGGAGGGGAGGGGAGGGGAGAP (SEQ ID NO: 82). Any one of these spacers isinserted between Naglu and IGF-II mutein, optionally via the AscI siteson the constructs.

An exemplary rigid linker which comprises multiple prolines tocontribute to rigidity, has the following sequenceGGGSPAPTPTPAPTPAPTPAGGGPS (SEQ ID NO: 48),GAPGGGSPAPTPTPAPTPAPTPAGGGPSGAP (SEQ ID NO: 49),GGGSPAPAPTPAPAPTPAPAGGGPS (SEQ ID NO: 50), orGAPGGGSPAPAPTPAPAPTPAPAGGGPSGAP (SEQ ID NO: 51), whereas an exemplaryhelical linker has the following sequence GGGSAEAAAKEAAAKEAAAKAGGPS (SEQID NO: 52), GAPGGGSAEAAAKEAAAKEAAAKAGGPSGAP (SEQ ID NO: 53),GGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGG (SEQ ID NO: 54), orGAPGGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGGGAP (SEQ ID NO: 55). Any one ofthese spacers is inserted between Naglu and IGF-II mutein, optionallyvia the AscI sites on the constructs.

Additional spacers can be generated using codon optimization usingtechnology developed by DNA 2.0 (Menlo Park, Calif.). The spacerscontemplated include

(SEQ ID NO: 32) GGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGPS, (SEQ ID NO: 33)GAPGGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGPSGAP, (SEQ ID NO: 28)GGGGSGGGGSGGGGSGGGGSGGGPSGGGGSGGGPS, (SEQ ID NO: 29)GAPGGGGSGGGGSGGGGSGGGGSGGGPSGGGGSGGGPSGAP, (SEQ ID NO: 30)GGGGSGGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGGSGGGGSGGGP S, (SEQ ID NO: 31)GAPGGGGSGGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGGSGGGGSGG GPSGAP,(SEQ ID NO: 34) GGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGPSGGGGSGGGGSGGGP S,(SEQ ID NO: 35) GAPGGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGPSGGGGSGGGGSGGGPSGAP, (SEQ ID NO: 36) GGGGSGGGGSGGGGSGGGGSGGGPS, (SEQ ID NO: 37)GAPGGGGSGGGGSGGGGSGGGGSGGGPSGAP, (SEQ ID NO: 38)GGGGSGGGGSAAAASGGGGSGGGPS, (SEQ ID NO: 39)GAPGGGGSGGGGSAAAASGGGGSGGGPSGAP, (SEQ ID NO: 40)GGGGSGGGGSAAAASGGGGSGGGGSAAAASGGGGSGGGGSAAAASGGGP S, (SEQ ID NO: 41)GAPGGGGSGGGGSAAAASGGGGSGGGGSAAAASGGGGSGGGGSAAAASGG GPSGAP,(SEQ ID NO: 42) GGGGSGGGGSAAAASGGGPSGGGGSAAAASGGGPSGGGGSAAAASGGGP S,(SEQ ID NO: 43) GAPGGGGSGGGGSAAAASGGGPSGGGGSAAAASGGGPSGGGGSAAAASGGGPSGAP, (SEQ ID NO: 67) GGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGPS,(SEQ ID NO: 68) GAPGGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGPSGAP,(SEQ ID NO: 63) GGGGAGGGGAGGGGAGGGGAGGGPSGGGGAGGGPS, (SEQ ID NO: 64)GAPGGGGAGGGGAGGGGAGGGGAGGGPSGGGGAGGGPSGAP, (SEQ ID NO: 65)GGGGAGGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGGAGGGGAGGGP S, (SEQ ID NO: 66)GAPGGGGAGGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGGAGGGGAGG GPSGAP,(SEQ ID NO: 69) GGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGPSGGGGAGGGGAGGGP S,(SEQ ID NO: 70) GAPGGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGPSGGGGAGGGGAGGGPSGAP, (SEQ ID NO: 71) GGGGAGGGGAGGGGAGGGGAGGGPS, (SEQ ID NO: 72)GAPGGGGAGGGGAGGGGAGGGGAGGGPSGAP, (SEQ ID NO: 73)GGGGAGGGGAAAAASGGGGAGGGPS, (SEQ ID NO: 74)GAPGGGGAGGGGAAAAASGGGGAGGGPSGAP, (SEQ ID NO: 75)GGGGAGGGGAAAAASGGGGAGGGGAAAAASGGGGAGGGGAAAAASGGGP S, (SEQ ID NO: 76)GAPGGGGAGGGGAAAAASGGGGAGGGGAAAAASGGGGAGGGGAAAAASGG GPSGAP,(SEQ ID NO: 77) GGGGAGGGGAAAAASGGGPSGGGGAAAAASGGGPSGGGGAAAAASGGGP S,(SEQ ID NO: 78) GAPGGGGAGGGGAAAAASGGGPSGGGGAAAAASGGGPSGGGGAAAAASGGGPSGAP, (SEQ ID NO: 87) GGGGPAPGPGPAPGPAPGPAGGGPS, (SEQ ID NO: 88)GAPGGGGPAPGPGPAPGPAPGPAGGGPGGAP, (SEQ ID NO: 89)GGGGPAPAPGPAPAPGPAPAGGGPS, and (SEQ ID NO: 90)GAPGGGGPAPAPGPAPAPGPAPAGGGPGGAP.

Any one of these spacers is inserted between Naglu and IGF-II mutein,optionally via the AscI sites on the constructs.

In certain embodiments if the BM-40 extracellular matrix protein signalpeptide sequence (Nischt et al., Eur J. Biochem 200:529-536, 1991) isused, the Naglu in the construct does not comprise its own signalpeptide sequence. The spacer is inserted between the Naglu sequence andthe IGF-II mutein sequence (e.g., IGF2 8-67 R37A). An exemplary BM-40signal peptide sequence is MRAWIFFLLCLAGRALA (SEQ ID NO: 8). A GAPpeptide may be added to the spacer to facilitate cloning and addition ofan AscI cloning site. In certain embodiments, if the native Naglu signalpeptide sequence (Weber et al., Hum Mol Genet. 5:771-777, 1996) is used,the Naglu is full-length Naglu and the spacer is inserted between thefull-length Naglu and the IGF-II mutein sequence (e.g., IGF2 8-67 R37A).A GAP peptide may be added to the spacer to facilitate cloning andaddition of an AscI cloning site.

In exemplary constructs, the human Naglu has been “codon optimized”using DNA 2.0 technology. It is contemplated that the Naglu comprisesamino acids 1-743 or amino acids 24-743 of human Naglu. In an exemplaryconstruct, the spacer optionally comprises a GAP spacer (AscIrestriction enzyme site used for cloning) or any of the followingsequences:

(SEQ ID NO: 22) EFGGGGSTR, (SEQ ID NO: 9) GAP, (SEQ ID NO: 12) GGGGS,(SEQ ID NO: 23) GPSGSPG, (SEQ ID NO: 24) GPSGSPGT, (SEQ ID NO: 25)GPSGSPGH, (SEQ ID NO: 26) GGGGSGGGGSGGGGSGGGGSGGGPST, (SEQ ID NO: 27)GGGGSGGGGSGGGGSGGGGSGGGPSH, (SEQ ID NO: 28)GGGGSGGGGSGGGGSGGGGSGGGPSGGGGSGGGPS, (SEQ ID NO: 29)GAPGGGGSGGGGSGGGGSGGGGSGGGPSGGGGSGGGPSGAP, (SEQ ID NO: 30)GGGGSGGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGGSGGGGSGGGP S, (SEQ ID NO: 31)GAPGGGGSGGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGGSGGGGSGG GPSGAP,(SEQ ID NO: 32) GGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGPS, (SEQ ID NO: 33)GAPGGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGPSGAP, (SEQ ID NO: 34)GGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGPSGGGGSGGGGSGGGP S, (SEQ ID NO: 35)GAPGGGGSGGGGSGGGGSGGGPSGGGGSGGGGSGGGPSGGGGSGGGGSGG GPSGAP,(SEQ ID NO: 36) GGGGSGGGGSGGGGSGGGGSGGGPS, (SEQ ID NO: 37)GAPGGGGSGGGGSGGGGSGGGGSGGGPSGAP, (SEQ ID NO: 38)GGGGSGGGGSAAAASGGGGSGGGPS, (SEQ ID NO: 39)GAPGGGGSGGGGSAAAASGGGGSGGGPSGAP, (SEQ ID NO: 40)GGGGSGGGGSAAAASGGGGSGGGGSAAAASGGGGSGGGGSAAAASGGGP S, (SEQ ID NO: 41)GAPGGGGSGGGGSAAAASGGGGSGGGGSAAAASGGGGSGGGGSAAAASGG GPSGAP,(SEQ ID NO: 42) GGGGSGGGGSAAAASGGGPSGGGGSAAAASGGGPSGGGGSAAAASGGGP S,(SEQ ID NO: 43) GAPGGGGSGGGGSAAAASGGGPSGGGGSAAAASGGGPSGGGGSAAAASGGGPSGAP, (SEQ ID NO: 44) GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPGPS,(SEQ ID NO: 45) GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPGPS GAP,(SEQ ID NO: 46) GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTGP S,(SEQ ID NO: 47) GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTGPSGAP, (SEQ ID NO: 48) GGGSPAPTPTPAPTPAPTPAGGGPS, (SEQ ID NO: 49)GAPGGGSPAPTPTPAPTPAPTPAGGGPSGAP, (SEQ ID NO: 50)GGGSPAPAPTPAPAPTPAPAGGGPS, (SEQ ID NO: 51)GAPGGGSPAPAPTPAPAPTPAPAGGGPSGAP, (SEQ ID NO: 52)GGGSAEAAAKEAAAKEAAAKAGGPS, (SEQ ID NO: 53)GAPGGGSAEAAAKEAAAKEAAAKAGGPSGAP, (SEQ ID NO: 54)GGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGG, (SEQ ID NO: 55)GAPGGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGGGAP, (SEQ ID NO: 56)GGGGSGGGGSGGGGS, (SEQ ID NO: 57) GAPGGGGSGGGGSGGGGSGAP, (SEQ ID NO: 58)GGGGSGGGGSGGGGSGGGGS, (SEQ ID NO: 59) GAPGGGGSGGGGSGGGGSGGGGSGAP,(SEQ ID NO: 60) GGGGA, (SEQ ID NO: 61) GGGGAGGGGAGGGGAGGGGAGGGPST,(SEQ ID NO: 62) GGGGAGGGGAGGGGAGGGGAGGGPSH, (SEQ ID NO: 63)GGGGAGGGGAGGGGAGGGGAGGGPSGGGGAGGGPS, (SEQ ID NO: 64)GAPGGGGAGGGGAGGGGAGGGGAGGGPSGGGGAGGGPSGAP, (SEQ ID NO: 65)GGGGAGGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGGAGGGGAGGGP S, (SEQ ID NO: 66)GAPGGGGAGGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGGAGGGGAGG GPSGAP,(SEQ ID NO: 67) GGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGPS, (SEQ ID NO: 68)GAPGGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGPSGAP, (SEQ ID NO: 69)GGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGPSGGGGAGGGGAGGGP S, (SEQ ID NO: 70)GAPGGGGAGGGGAGGGGAGGGPSGGGGAGGGGAGGGPSGGGGAGGGGAGG GPSGAP,(SEQ ID NO: 71) GGGGAGGGGAGGGGAGGGGAGGGPS, (SEQ ID NO: 72)GAPGGGGAGGGGAGGGGAGGGGAGGGPSGAP, (SEQ ID NO: 73)GGGGAGGGGAAAAASGGGGAGGGPS, (SEQ ID NO: 74)GAPGGGGAGGGGAAAAASGGGGAGGGPSGAP, (SEQ ID NO: 75)GGGGAGGGGAAAAASGGGGAGGGGAAAAASGGGGAGGGGAAAAASGGGP S, (SEQ ID NO: 76)GAPGGGGAGGGGAAAAASGGGGAGGGGAAAAASGGGGAGGGGAAAAASGG GPSGAP,(SEQ ID NO: 77) GGGGAGGGGAAAAASGGGPSGGGGAAAAASGGGPSGGGGAAAAASGGGP S,(SEQ ID NO: 78) GAPGGGGAGGGGAAAAASGGGPSGGGGAAAAASGGGPSGGGGAAAAASGGGPSGAP, (SEQ ID NO: 79) GGGGAGGGGAGGGGA, (SEQ ID NO: 80)GAPGGGGAGGGGAGGGGAGAP, (SEQ ID NO: 81) GGGGAGGGGAGGGGAGGGGA,(SEQ ID NO: 82) GAPGGGGAGGGGAGGGGAGGGGAGAP, (SEQ ID NO: 83)GGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGPS [or (GGGGA)8GGGPS],(SEQ ID NO: 84) GGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGPSH[or (GGGGA)8GGGPSH], (SEQ ID NO: 85)GGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGPS [or (GGGGA)9GGGPS],(SEQ ID NO: 86) GGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGPSH [or (GGGGA)9GGGPSH], (SEQ ID NO: 87) GGGGPAPGPGPAPGPAPGPAGGGPS,(SEQ ID NO: 88) GAPGGGGPAPGPGPAPGPAPGPAGGGPGGAP, (SEQ ID NO: 89)GGGGPAPAPGPAPAPGPAPAGGGPS, and (SEQ ID NO: 90)GAPGGGGPAPAPGPAPAPGPAPAGGGPGGAP.

The above spacers are optionally “codon optimized” using DNA 2.0technology.

Any of the IGF-II muteins described herein, which are optionally “codonoptimized” using DNA 2.0 technology, are useful in the presentconstructs. In exemplary constructs, the IGF-II mutein is afurin-resistant IGF-II mutein, IGF2 Δ8-67 R37A.

Example 2—Expression and Purification of Constructs

Constructs comprising the above spacers, the Naglu enzyme and the IGF-IItargeting peptide are made and recombinantly expressed. In certainembodiments, the constructs comprise a signal peptide. Exemplary signalpeptides include, for example and not for limitation, the Naglu signalpeptide comprising amino acids 1-23 of full-length Naglu (Weber et al.,Hum Mol Genet 5:771-777, 1996) or a signal peptide derived from theBM-40 extracellular matrix protein (Nischt et al., Eur J Biochem200:529-536, 1991).

DNA encoding the Naglu sequence, the IGF-II mutein and the spacerpeptide are inserted into an appropriate expression vector, such as thepEE and pXC GS expression vectors (Lonza Biologics, Berkshire, UK) andthe pC3B (BioMarin, in-house) expression vector. An AscI restrictionsite (ggcgcgcc (SEQ ID NO: 570)) can be inserted into the vector to aidin cloning the therapeutic fusion proteins described herein.

An exemplary construct comprises the full-length Naglu sequence (FIG. 1and FIG. 2), including signal peptide, a spacer peptide (FIG. 3) and anIGF-II peptide comprising residues 8-67 and having an Ala amino acidsubstitution at residue Arg-37, R37A (FIGS. 1 and 2), that confers furinresistance to the IGF-II peptide.

Various linker or spacer sequences described in Example 1, connectingthe Naglu and IGF-II peptide, are initially evaluated using a transientexpression system. GILT-tagged Naglu plasmids (pXC17.4, Lonza) aretransfected into suspension CHOK1SV GS KO cells (Lonza). 15 μg ofplasmid DNA is transfected into 10⁶ cells using electroporation. Mediais completely exchanged at 24 hours post-transfection. The transfectedcells are seeded in shaker flasks at 1.5×10⁶ cells/ml without selection.Cell growth, viability, titer and specific productivity are determinedas the cells are grown at 30° C. for up to 14 days.

GILT-tagged Naglu plasmids are transfected into suspension CHOK1SV cells(Lonza). The cells are grown in CDCHO media (Invitrogen) with 6 mMglutamine in shake flasks at 37° C. and 8% CO₂. 30 μg of linearizedplasmid DNA in 1×10⁷ cells is transfected into the cells usingelectroporation. The cells are plated at 5000 cells/well in CDCHOmedia+40 μM MSX 48 hours after transfection. The plates are incubated at37° C. and 8% CO₂ for approximately 4-6 weeks to identify clonal growth.The colonies are then screened by the 4MU activity assay for Naglu (seeExample 3) and the highest expressing colonies are transferred to 24well plates in CDCHO media+40 μM MSX, and then continued to passage thehighest expressing clones to 6 well plates, then to shake flasks toidentify the highest expressing clones to produce the GILT-tagged Naglufusion proteins.

Purification is carried out using standard protein purificationtechniques. For example, in an exemplary purification method, startingmaterial of mammalian cell culture supernatant, as described above, isthawed from storage at −80° C. The material is adjusted with NaCl toreach a final concentration of 1M, followed by 0.2 μm sterilefiltration.

The filtered material is loaded onto a butyl hydrophobic interactioncolumn, pre-equilibrated with butyl load buffer (20 mM Tris, 1 M NaCl,pH 7.5). The bound materials are eluted with a linear gradient over 10column volumes, using butyl elution buffer (20 mM Tris, pH 7.5). Samplesfrom the elution peaks are pooled, buffer exchanged into 20 mM Tris, pH7.5, and loaded onto a Q anion exchange column. Bound proteins are theneluted with a linear gradient (10 column volumes) using Q elution buffer(20 mM Tris, 1 M NaCl, pH 7.5). Purified samples are then bufferexchanged using centrifugal spin concentrators and sterile-filtered forstorage.

Construction, Expression, Production, Purification and Formulation of anExemplary Naglu Fusion Protein: Naglu-(GGGGS)₄GGGPS-IGF-II (SEQ ID NO:568).

A DNA construct encoding Naglu-(GGGGS)₄GGGPS-IGF-II (SEQ ID NO: 568) wasgenerated by standard recombinant DNA methods. Naglu corresponds toamino acids 1-743 of full-length human Naglu and IGF-II corresponds tothe IGF-II mutein comprising amino acids 8-67 of mature human IGF-IIwith the R37A amino acid substitution that confers furin-resistance.CHOK1SV cells were transfected with the DNA construct, and a stableGILT-tagged Naglu-IGF-II fusion protein expressing clone was isolated asdescribed above.

Cells expressing Naglu-(GGGGS)₄GGGPS-IGF-II (SEQ ID NO: 568) were grownin a bioreactor, and the Naglu fusion protein was purified from theculture medium as follows. The harvest was salt-adjusted to 1 M NaCl,then loaded onto a Butyl Sepharose 4 FF column. The Naglu fusion proteinwas salt-eluted from the Butyl Sepharose 4 FF column, collected anddialyzed, and then loaded onto a Heparin Sepharose 6 FF column. TheNaglu fusion protein was collected in the flow-through fraction, andloaded onto a Q Sepharose HP column. The Naglu fusion protein wassalt-eluted from the Q Sepharose HP column, concentrated, and thenpolished by preparative Sephacryl S300 size exclusion chromatography.

Using this purification procedure, a highly purified, enzymaticallyactive Naglu fusion protein, Naglu-(GGGGS)₄GGGPS-IGF-II (SEQ ID NO:568), was produced. The purified Naglu fusion protein was formulated at20 mg/mL in artificial CSF (1 mM sodium phosphate, 148 mM sodiumchloride, 3 mM potassium chloride, 0.8 mM magnesium chloride, 1.4 mMcalcium chloride, pH 7.2).

Construction, Expression, Production, Purification and Formulation ofExemplary Naglu Fusion Proteins.

DNA constructs encoding Naglu-(GGGGA)₄GGGPS-IGF-II (SEQ ID NO: 569),Naglu-Rigid-IGF-II (SEQ ID NO: 566), Naglu-Helical-IGF-II (SEQ ID NO:567), and Naglu-XTEN-IGF-II (SEQ ID NO: 565) were generated by standardrecombinant DNA methods. Naglu corresponds to amino acids 1-743 offull-length human Naglu and IGF-II corresponds to the IGF-II muteincomprising amino acids 8-67 of mature human IGF-II with the R37A aminoacid substitution that confers furin-resistance. Exemplary Rigid,Helical and XTEN linkers are described in Example 1. CHOK1SV cells weretransfected with the DNA constructs, and stable GILT-tagged Naglu-IGF-IIfusion protein expressing clones were isolated as described above.

Cells expressing the Naglu-IGF-II fusion proteins were grown in abioreactor. In typical fed-batch production runs (10˜16 days),Naglu-IGF-II constructs with the various linkers all reached titersabove 30 mg/L with high cell viability above 80%.

The Naglu fusion proteins were purified from the culture medium asdescribed above. Using this purification procedure, enzymatically activeuntagged Naglu and Naglu-IGF-II fusion proteins, such asNaglu-Rigid-IGF-II (SEQ ID NO: 566), Naglu-Helical-IGF-II (SEQ ID NO:567), Naglu-XTEN-IGF-II (SEQ ID NO: 565) and Naglu-(GGGGA)₄GGGPS-IGF-II(SEQ ID NO: 569), were purified to ˜99% purity, as determined byreverse-phase HPLC. The purified untagged Naglu and Naglu fusionproteins were formulated at 20 mg/mL in artificial CSF (1 mM sodiumphosphate, 148 mM sodium chloride, 3 mM potassium chloride, 0.8 mMmagnesium chloride, 1.4 mM calcium chloride, pH 7.2).

It is contemplated that fusion proteins as described herein thatdemonstrate higher levels of recombinant expression of active proteinand/or increased enzymatic activity compared to fusion proteinscomprising a different spacer peptide may be used in furtherexperimentation, such as activity assays, binding assays, uptake assaysand in vivo activity assays as described further below.

Example 3—Activity Assays

To determine the enzymatic activity of the Naglu fusion proteins, an invitro Naglu activity assay is carried out using a fluorescent labeledsynthetic substrate.

Materials used in the assay include:4-Methylumbelliferyl-N-acetyl-a-D-glucosaminide (4MU-NaGlu Substrate)(Calbiochem, Cat#474500) prepared to final 20 mM concentration in 10%DMSO in the assay buffer (0.2 M Sodium Acetate, with or without 1 mg/mlBSA, and 0.005% Tween 20, pH 4.3-4.8) and stored at −80° C. Stocksolution of 4-Methylumbelliferone (4-MU Standard) (Sigma, Cat# M1381) isprepared at 10 mM in DMSO and stored at −20° C. in small aliquots. ArhNaglu-His6 control (0.5 mg/ml, R&D Systems, Cat #7096-GH) is dilutedto 10 μg/ml in 25 mM Tris, 125 mM NaCl, 0.001% Tween 20, pH7.5 andstored at −80° C. in small aliquots.

On a clear 96 well dilution plate (Granger), 2× serial dilutions ofstandards in Dilution Buffer (1×PBS, with or without 1 mg/ml BSA, 0.005%Tween 20, pH 7.4 are used, from 200 μM to 1.563 μM plus one blank. On aclear dilution plate, samples are prepared in several dilutions (inDilution Buffer) to ensure that they are within the standard curve.

10 μl of standards (200 μM to 1.563 μM), control and working samples aretransferred to a black non-treated polystyrene 96 well plate (Costar,Cat#3915). 75 μl of substrate (2 mM) is added to each well, followed byincubation for 30 minutes at 37° C. The reaction is then quenched byaddition of 200 μl of stop buffer (0.5 M Glycine/NaOH, pH 10.7). Theplates are read on an Ex355 Em460 with 455 cut off on a 96-wellfluorescent plate reader.

Using this assay, exemplary Naglu fusion proteins, includingNaglu-(GGGGS)₄GGGPS-IGF-II (SEQ ID NO: 568), Naglu-(GGGGA)₄GGGPS-IGF-II(SEQ ID NO: 569), Naglu-Rigid-IGF-II (SEQ ID NO: 566),Naglu-Helical-IGF-II (SEQ ID NO: 567), and Naglu-XTEN-IGF-II (SEQ ID NO:565), were shown to have enzymatic activity in vitro, with specificactivities toward the synthetic 4MU-Naglu substrate ranging from˜175,000 to 220,000 nmol/hr/mg. The enzymatic activity of the Naglufusion proteins was comparable to that of the untagged Naglu protein(˜190,000 nmol/hr/mg). Enzymatic activity data for exemplary Naglufusion proteins is provided in Table 1.

TABLE 2 Activity of Naglu Fusion Proteins Naglu¹ Linker² Sp. Act.³ IC₅₀⁴ K_(uptake) ⁵ t_(1/2) ⁵ Untagged Naglu — 190,000 — — 9.7Naglu-(GGGGS)₄GGGPS-IGF-II 36 190,000 0.27, 0.23 5.4 NDNaglu-(GGGGA)₄GGGPS-IGF-II 71 220,000 0.36 6.3 ND Naglu-Rigid-IGF-II 51190,000 0.23 2.4 9.5 Naglu-Helical-IGF-II 55 175,000 0.25 2.3 9.4Naglu-XTEN-IGF-II 47 170,000 0.24 3.7 ND ¹Untagged Naglu and Naglufusion proteins were constructed, expressed and purified as described inExample 2; exemplary Rigid, Helical and XTEN linkers are described inExample 1 ²SEQ ID NO: of linkers in the Naglu fusion proteins tested inExamples 3 to 5 ³Specific activity (nmol/hr/mg) for Naglu proteins wasmeasured as described in Example 3 ⁴IC₅₀ for Naglu proteins for IGF2Rcompetitive binding was measured as described in Example 4 ⁵K_(uptake)and half life (t_(1/2)) for Naglu proteins in MPS-IIIB fibroblasts weremeasured as described in Example 5

Example 4—Binding Assays

Binding assays to determine binding of the Naglu fusion proteins toIGF-I, IGF-II and insulin receptors are carried out generally asdescribed in US 20120213762. Briefly, fusion protein constructs aretested for binding affinity for the insulin receptor in an assaymeasuring the competition of biotinylated insulin binding to plate-boundinsulin. An insulin receptor binding assay is conducted by competinginsulin, IGF-II, and fusion protein with biotinylated-insulin binding tothe insulin receptor (Insulin-R).

Specifically, white Reacti-Bind plates are coated with Insulin-R at aconcentration of 1 μg/well/100 μl (38.4 nM). The coated plates areincubated over night at room temperature, then washed 3× with washingbuffer (300 μl/well). The plates are then blocked with blocking buffer(300 μl/well) for 1 hour. The washing steps are repeated and any traceof solution in the plates taken out. Biotinylated-insulin is mixed at 20nM with different concentrations of insulin, IGF-II, or fusion protein,by serial dilutions. 100 μl of diluted Insulin, IGF-II, or Naglu fusionprotein in 20 nM Insulin-biotin are added into the coated plates and theplates are incubated at room temperature for 2 hours. The plates arethen washed 3 times with washing buffer. 100 μl of strepavidin-HRPworking solution (50 μl strepavidin-HRP in 10 ml blocking buffer) isadded into the plates and the plates are incubated at room temperaturefor 30 minutes. 100 μl of Elisa-Pico working solution containingElisa-Pico chemiluminescent substrate is added and the chemiluminescenceis measured at 425 nm.

IGF2R Competitive Binding Assay

To measure the ability of the Naglu fusion protein constructs to bind tothe IGF-II receptor a competitive binding assay is carried out. Afragment of the IGFIIR involved with IGF-II binding (domains 10-13,named protein 1288) is coated onto 96-well plates. Biotinylated IGF-IIis incubated with the receptor in the presence of increasing amounts ofcompetitors: either control IGF-II (non-biotinylated), or fusion proteinsample (containing an IGF-II-derived GILT epitope tag). Receptor-boundbiotinylated IGF-II is detected with streptavidin conjugated tohorseradish peroxidase (HRP) and a chemiluminescent HRP substrate. Theability of the fusion protein to inhibit binding of biotinylated IGF-IIto the IGFIIR is calculated from inhibition curves and reported as anIC₅₀ value (concentration required to achieve 50% binding inhibition).

For the assay, IGFIIR is coated in a white Reacti-bind plate (Pierce,Cat#437111) at 0.5 μg/well in a volume of 100 μl (69.6 nM/per well) incoating buffer. The plate is sealed and incubated overnight at roomtemperature. The plate is then washed 3× with wash buffer, blocked withblocking buffer and then washed again 3× with wash buffer (300 μl/well).

Next, 8 nM IGF-II-biotin is mixed with different concentrations ofcompetitors (IGF-II (non-biotinylated), Reference Protein, or Naglufusion protein samples, and added into an IGFIIR-coated plate in 2×serial dilutions.

The plate is incubated at room temperature for 2 hours, followed bywashing the plate 3× with wash buffer. Streptavidin-HRP is prepared inblocking buffer (1:200 dilution), and 100 μl/well added to the plate.IGF-II-Biotin binding activity is detected via streptavidin-HRP usingPico-Elisa reagents. Briefly, the prepared Pico-Elisa working solutionis added per well (100 μl/well), and incubated at room temperature for 5minutes with gentle rocking, then the chemiluminescence at 425 nm ismeasured.

The IC₅₀ of the samples are calculated using the percent IGF-II-BiotinBound for each concentration of inhibitor.

Using this competitive IGFIIR binding assay, an exemplary Naglu fusionproteins, including Naglu-(GGGGS)₄GGGPS-IGF-II (SEQ ID NO: 568),Naglu-(GGGGA)₄GGGPS-IGF-II (SEQ ID NO: 569), Naglu-Rigid-IGF-II (SEQ IDNO: 566), Naglu-Helical-IGF-II (SEQ ID NO: 567), and Naglu-XTEN-IGF-II(SEQ ID NO: 565), were shown to have an IC₅₀ value of 0.23-0.36 nM.Untagged Naglu protein had no detectable binding in this assay. IGF2Rcompetitive binding data for exemplary Naglu fusion proteins is providedin Table 1.

Example 5—Uptake Assays

To measure the ability of a Lysosomal Storage Disease enzyme to entercells via receptor-mediated endocytosis an uptake assay is carried outwhich measures enzyme uptake using the CI-MPR receptor in rat myoblastL6 cells or in human MPS IIIB fibrobalsts. Mannose-6-phosphate (M6P) andIGF-II are used as inhibitors to determine the site of binding to theCI-MPR receptor. Data is collected to generate a saturation curve forenzyme uptake and determine the kinetic parameter, K_(uptake), of theprocess.

Prior to the uptake assay (24 hours), L6 cells (L6 Rat Myoblasts,ATCC#CRL-1458) or human MPS IIIB fibroblasts are plated at a density of1×10⁵ cells per well in 24-well plates (VWR #62406-183) and seeded 0.5ml per well. On the morning of assay, enzyme is mixed with uptake media(1 L DMEM, 1.5 g Sodium Bicarbonate. 0.5 g Bovine Serum Albumin, 20 mlof L-glutamine (200 mM (Gibco #25030-081)), 20 ml of 1M of HEPES (Gibco#1563080)) (20 mM final), pH 7.2) in a tissue culture hood. Enzymeamounts may range from 2-500 nM. The final volume of uptake media+enzymeis 0.5 ml per well. M6P (5 mM final concentration) and/or IGF-II (2.4 μMor 18 μg/ml final concentration) are added to appropriate samples. Foruptake inhibition, 18 μl IGF-II stock (1 mg/ml, 133.9 μM) is added permL of uptake media.

Growth media is aspirated from cells and 0.450 ml of enzyme in uptakebuffer added to each well. Note time and return cells to incubator for18 hours. Plate is removed from incubator and uptake buffer aspiratedoff cells. Plates are washed 4× by addition of 0.5 ml Dulbecco's PBS andaspirating off. 200 μl of CelLytic M lysis buffer (Sigma) is added tothe plates and shaken at room temperature for 20-30 minutes. Lysate isremoved from cells and stored in a tape-covered clear 96-well plate(VWR) at −80° C. until ready to assay.

For the enzyme assay, 5 μl of each lysate is added in duplicate byadding to 15 μl of enzyme reaction mix (e.g., Naglu+4MU assays) in black96-well plate (VWR) (see above) and enzyme/units/ml/hr determined ineach lysate.

For the lysate protein assay, 10 μl of each lysate in duplicate areassayed using a Pierce BCA protein Assay kit according to manufacturer'sinstructions. To measure absorbance, absorbance is read at 562 nm with aplate reader (BMG FluoStar Optima Plate reader) and ug/ml concentrationdetermined.

For each enzyme load, uptake is units of enzyme activity/mg lysate. Todetermine uptake, the enzyme units/ml are divided by protein ug/ml andmultiplied by 1000 (uptake from blank wells subtracted). Results of theassays with or without inhibitors are compared to determine receptoruptake specificity.

For saturation curves, 10 enzyme load concentrations ranging from0.2-100 nM are used to generate a saturation curve using the assaysdescribed above.

Using this assay, an exemplary Naglu fusion protein,Naglu-(GGGGS)₄GGGPS-IGF-II (SEQ ID NO: 568), was shown to have aK_(uptake) of 7-9 nM in MPS-IIIB fibroblasts.

Alternatively, prior to the uptake assay (24 hours), L6 cells or humanMPS IIIB fibroblasts are plated at a density of 1×10⁵ cells per 0.5 mlper well in the 24-well plates. Enzyme samples at 1.6˜50 nM are preparedin uptake media: 1 L DMEM, 1.5 g Sodium Bicarbonate. 0.5 g Bovine SerumAlbumin, 20 ml of 200 mM L-glutamine and 20 ml of 1M HEPES, pH7.2. Foruptake inhibition, M6P (up to 5.0 mM final) and/or IGF-II (up to 1.0 μMfinal) are added to appropriate samples.

Growth media is aspirated from cells and replaced by 0.5 ml of theenzyme preparation in uptake buffer per well. After 4-hour incubation,plates are washed 2 times with 0.5 ml Dulbecco's PBS. 100 μl of M-PERlysis buffer (Pierce) is added to the plates and shaken at roomtemperature for 10 minutes. Lysate is stored at −80° C. until ready toassay.

For the enzyme assay, 10 μl of each lysate is added in duplicate to theblack 96-well plate (see above).

For the lysate protein assay, 10 μl of each lysate in duplicate areassayed using a Pierce BCA Protein Assay Kit according to manufacturer'sinstructions. Absorbance is read at 562 nm with a plate reader (BMGFluoStar Optima Plate reader) and μg/ml concentration determined usingBSA as a standard.

For each enzyme load, uptake is expressed as nmoles of 4-MU liberated in30 minutes. For saturation curves, enzyme concentrations ranging from1.6-50 nM are used to generate a saturation curve using the assaysdescribed above.

Cellular stability of the Naglu fusion proteins was determined bymonitoring intracellular Naglu activity over the period of −8 days.Human MPS IIIB fibroblasts plated at a density of 1×10⁵ cells per wellin 24-well plates (VWR #62406-183) were treated with Naglu fusionprotein at 20 nM final concentration for 4 hours. After 4-hourincubation, cells were switched to growth media without Naglu fusionprotein. For each time point (4 hours, 28 hours, 4 days, 6 days & 8days), cells were lysed in 100 μl of M-PER lysis buffer (Pierce) at roomtemperature for 10 minutes, and assayed for enzyme activity using a 4-MUlabeled substrate. Reduction of Naglu activity over the 8-day sampleperiod can be fit to first-order kinetics to approximate a cellularhalf-life of the protein.

Using this assay, exemplary Naglu fusion proteins, includingNaglu-(GGGGS)₄GGGPS-IGF-II (SEQ ID NO: 568), Naglu-(GGGGA)₄GGGPS-IGF-II(SEQ ID NO: 569), Naglu-Rigid-IGF-II (SEQ ID NO: 566),Naglu-Helical-IGF-II (SEQ ID NO: 567), and Naglu-XTEN-IGF-II (SEQ ID NO:565), were shown to be internalized into MPS IIIB fibroblasts withK_(uptake) values of ˜2.3-6.3 nM. Untagged Naglu protein, in contrast,was not taken up by the cells under these experimental conditions.Furthermore, the observed uptake of Naglu fusion protein was inhibitedby IGF-II, but not by M6P. After uptake, exemplary Naglu fusion proteinswere found to be stable with an estimated half-life of ˜9.5 days, basedon enzymatic activity (4-MU substrate) measured in cell lysates. Uptakeand half-life data for exemplary Naglu fusion proteins is provided inTable 1.

Example 6—In Vivo Naglu Fusion Protein Activity

To determine the activity of Naglu fusion proteins in vivo, the fusionproteins are administered to Naglu knock-out animals (see Li et al.,Proc Natl Acad Sci USA 96:14505-510, 1999). Naglu knockouts present withlarge amounts of heparan sulfate in the brain, liver and kidney,increase of beta-hexosaminidase activity and lysosomal-associatedmembrane protein 2 (LAMP-2) staining in brain, and elevation ofgangliosides in brain.

Activity and biodistribution of the exogenous enzyme are determinedafter 4 ICV (intracerebroventricular) injections over a two week period(100 μg/injection) of recombinant human (rh) Naglu-IGF2. A permanentcannulae is implanted in the mouse (n=12/gp, 8-12 wks old at start) andadjusted to cover those mice whose cannulae are not in the ventricle.Endpoint measurements include Naglu biodistribution, reduction of GAG,e.g., heparan sulfate, storage in the lysosomes of brain cells, andactivation of astrocytes and microglia. Levels of various lysosomal orneuronal biomarkers (Ohmi et al., PLoS One 6:e27461, 2011) measured intreated and control groups levels include, but are not limited to,Lysosomal-associated membrane protein 1 (LAMP-1), LAMP-2, glypican 5,Naglu-specific non-reducing ends (NREs) of heparan sulfate,gangliosides, cholesterol, Subunit c of Mitochondrial ATP Synthase(SCMAS), ubiquitin, P-GSK3beta, beta amyloid, P-tau(phosphorylated-Tau), GFAP (astrocyte activation) and CD68 (microglialactivation).

Additional experiments to determine survival and behavioral analysis arecarried out using mice receiving 4 ICV injections over a two week periodof rhNaglu-IGF2 (n=12/gp, 5 months old at start, 100 μg/injection).Endpoints to be measured include survival time, open field activity,Naglu biodistribution, reduction of GAG, e.g., heparan sulfate, storagein lysosomes, levels of lysosomal or neuronal biomarkers, such asLAMP-1, LAMP-2, glypican 5, gangliosides, cholesterol, SCMAS, ubiquitin,P-GSK3beta, beta amyloid, P-tau, GFAP and CD68.

Naglu knockout mice (Naglu −/−) having a mutation in exon 6 of the naglugene have been developed (Li et al., Proc Natl Acad Sci USA.96:14505-10, 1999). The exon 6 site was chosen because this is the siteof many mutations in humans. No Naglu activity is detected in homozygousmice, and there is reduced Naglu activity in heterozygotes. Naglu−/−mice have reduced survival times (6-12 months), and may have otherfunctional phenotypes like reduced activity levels. The effects of Naglufusion proteins on the Naglu −/− mice are assayed.

Naglu −/− mice (n=8) and 8 vehicle control Naglu −/− mice (n=8littermate heterozygotes) are administered 4 ICV doses (100 μgNaglu-IGF2/dose) over 2 weeks. At day −2, mice are anesthetized and theleft lateral ventricle cannulated. The mice are allowed to recover. Atdays 1, 5, 10 and 14, mice are anesthetized (Benedryl, 5 mg/kg IP) 15minutes prior to ICV dose. The ICV dose is infused via cannula, 5 μlvolume over 15 minutes, and mice are allowed to recover. On day 15, miceare sacrificed, exsanguinated and serum frozen. Brains are harvested andIR dye is injected into the cannula and the cannula imaged.

The following assays are carried out to determine the effects of Naglufusion proteins: body weight assessment, NIR imaging for cannulaplacement, assessment of Naglu-IGF2, GFAP, LAMP-1 and LAMP-2 levels inbrain using immunohistochemistry, biochemical assay for Naglu activity,β-hexosaminadase levels and activity, SensiPro assay to detectnon-reducing ends of accumulated glycosaminoglycans (GAGs) specific forMucopolysaccharidosis IIIb (MPS-IIIb) (WO 2010/078511A2), GM3ganglioside levels as measured by biochemical assay, as well asimmunostain for SCMAS, A-beta, glypican 5, CD68, GFAP and Naglu inmedial entorrhinal cortex (Li et al., supra).

Effective treatment with Naglu-IGF2 is expected to result in a decreasein levels of LAMP-1, LAMP-2, GFAP, CD68, SCMAS, A-beta, glypican 5,β-hexosaminadase, GM3 ganglioside, and MPS-IIIb-specific GAGs.

In Vivo Efficacy of Exemplary Naglu Fusion Proteins in a Mouse Model ofMPS IIIb.

Four ICV doses (100 μg/dose) of Naglu-IGF-II fusion protein, eitherNaglu-(GGGGS)₄GGGPS-IGF-II (SEQ ID NO: 568) or Naglu-Rigid-IGF-II (SEQID NO: 566), were administered over a two week period to Naglu −/− mice(n=8). Naglu −/− mice (n=8) and eight heterozygous or wild-typelittermates (n=8) were given vehicle alone as a control. At day −5, micewere anesthetized; the left lateral ventricle of the brain wascannulated. The mice were allowed to recover. On days 1, 5, 10 and 14,mice were anesthetized with inhaled isoflourane. Benadryl (5 mg/kg IP)was administered to each mouse 15 minutes prior to ICV dose to reduceany potential histamine release in response to the Naglu-IGF-IItreatment. The ICV dose was infused via the implanted cannula, 5 μlvolume over 15-20 minutes, and the mice were allowed to recover. At 1,7, 14, and 28 days following the final dose, mice were sacrificed.Brains were harvested and divided sagittally into 5 sections fordistribution to various assays.

The following assays were carried out to determine the effects ofNaglu-IGF-II fusion protein: immunohisochemical assessment of Naglu,LAMP-2, GFAP and CD68 levels in brain, biochemical assays for Naglu andbeta-hexosaminadase activity, SensiPro assay (Deakin et al.,Glycobiology 18:483, 2008; Lawrence et al., Nat Chem Biol. 8:197, 2012;Lawrence et al., J Biol Chem. 283:33674, 2008) to detect total heparansulfate and NREs of heparan sulfate specific for MucopolysaccharidosisIIIB (MPS-IIIB) (WO 2010/078511A2), and immunofluorescent staining forSCMAS, beta-amyloid (A-beta), p-Tau, P-GSK3beta, glypican 5, GFAP andCD68 in medial entorrhinal cortex (Li et al., supra).

When evaluated 24 hours after the final dose, treatment with eitherNaglu-(GGGGS)₄GGGPS-IGF-II (SEQ ID NO: 568) or Naglu-Rigid-IGF-II (SEQID NO: 566) fusion protein resulted in a marked increase in Naglu enzymeactivity, with a concomitant decrease in beta-hexosaminadase activityand levels of total heparan sulfate, Naglu-specific NREs of heparansulfate, and LAMP-2. Naglu enzyme was easily detectable in braintissues, not only in cortex, hippocampus, dentate gyrus and thalamus,but also in remote distal geographic locations, including amygdyla,perirhinal cortex and hypothalamus. Significant decreases in the levelsof CD68, SCMAS, beta-amyloid (A-beta), p-Tau, P-GSK3beta, and glypican 5were also observed in Naglu −/− brains upon treatment with Naglu-IGF-II.GFAP staining did not change by 24 hours post-last-dose.Immunohistochemistry demonstrated the presence of Naglu enzyme in manyareas of the brain, inside neurons and glial cells, co-localizing withLAMP-2.

Levels of heparan sulfate, Naglu-specific NREs, and beta-hexosaminidaseactivity continued to decrease over the 7, 14, and 28days-post-last-dose timepoints. At 28 days, all analytes were at or nearthe normal mouse control values.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the appended claims. The articles “a”, “an”,and “the” as used herein in the specification and in the claims, unlessclearly indicated to the contrary, should be understood to include theplural referents. Claims or descriptions that include “or” between oneor more members of a group are considered satisfied if one, more thanone, or all of the group members are present in, employed in, orotherwise relevant to a given product or process unless indicated to thecontrary or otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention also includes embodiments in which more than one, or all, ofthe group members are present in, employed in, or otherwise relevant toa given product or process. Furthermore, it is to be understood that theinvention encompasses variations, combinations, and permutations inwhich one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the claims is introduced into another claimdependent on the same base claim (or, as relevant, any other claim)unless otherwise indicated or unless it would be evident to one ofordinary skill in the art that a contradiction or inconsistency wouldarise. Where elements are presented as lists, e.g., in Markush group orsimilar format, it is to be understood that each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should it be understood that, in general, where the invention,or aspects of the invention, is/are referred to as comprising particularelements, features, etc., certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements, features, etc. For purposes of simplicity those embodimentshave not in every case been specifically set forth herein. It shouldalso be understood that any embodiment of the invention, e.g., anyembodiment found within the prior art, can be explicitly excluded fromthe claims, regardless of whether the specific exclusion is recited inthe specification.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one act,the order of the acts of the method is not necessarily limited to theorder in which the acts of the method are recited, but the inventionincludes embodiments in which the order is so limited. Furthermore,where the claims recite a composition, the invention encompasses methodsof using the composition and methods of making the composition. Wherethe claims recite a composition, it should be understood that theinvention encompasses methods of using the composition and methods ofmaking the composition.

All publications and patent documents cited in this application areincorporated by reference in their entirety to the same extent as if thecontents of each individual publication or patent document wereincorporated herein.

What is claimed:
 1. A nucleic acid encoding a targeted therapeuticfusion protein comprising (a) a lysosomal enzyme, (b) a peptide taghaving an amino acid sequence at least 70% identical to amino acids 8-67of mature human IGF-II, and (c) a spacer peptide located between theNaglu amino acid sequence and the IGF-II peptide tag, wherein the spacerpeptide comprises a sequence selected from (SEQ ID NO: 36)GGGGSGGGGSGGGGSGGGGSGGGPS, (SEQ ID NO: 47)GAPGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST GPSGAP,(SEQ ID NO: 51) GAPGGGSPAPAPTPAPAPTPAPAGGGPSGAP, (SEQ ID NO: 55)GAPGGGSPAEAAAKEAAAKEAAAKEAAAKEAAAKAPSGGGGAP, or (SEQ ID NO: 71)GGGGAGGGGAGGGGAGGGGAGGGPS.


2. The nucleic acid of claim 1, wherein the lysosomal enzyme has anamino acid sequence at least 85% identical to a humanα-N-acetylglucosaminidase (Naglu) protein.
 3. The nucleic acid of claim1, wherein the peptide tag is an N-terminal tag or a C-terminal tag. 4.The nucleic acid of claim 1, wherein the peptide tag is a C-terminaltag.
 5. The nucleic acid of claim 1, wherein the peptide tag comprisesamino acids 8-67 of mature human IGF-II.
 6. The nucleic acid of claim 4,wherein the peptide tag comprises a mutation at residue Arg37.
 7. Thenucleic acid of claim 5, wherein the mutation is a substitution ofalanine for arginine.
 8. The nucleic acid of claim 1, wherein thelysosomal enzyme is human α-N-acetylglucosaminidase (Naglu) protein, thepeptide tag comprises amino acids 8-67 of mature human IGF-II, and thepeptide tag comprises a mutation at residue Arg37.
 9. A cell containingthe nucleic acid of any of the preceding claims.
 10. A method ofproducing a targeted therapeutic fusion protein comprising culturing thecell of claim 9, wherein the culturing is performed under conditionsthat permit expression of the targeted therapeutic fusion protein.